Bispecific anti-vegf/anti-ang-2 antibodies

ABSTRACT

The present invention relates to bispecific antibodies against human VEGF and against human ANG-2, methods for their production, pharmaceutical compositions containing said antibodies, and uses thereof.

PRIORITY TO RELATED APPLICATION(S)

This application is a continuation of U.S. application Ser. No.16/534,295, filed Aug. 7, 2019 which is a continuation of U.S.application Ser. No. 15/603,124, filed May 23, 2017, which is acontinuation of U.S. application Ser. No. 14/192,213, filed Feb. 27,2014, now U.S. Pat. No. 9,708,396, which is a continuation of U.S.application Ser. No. 13/570,333, filed Aug. 9, 2012, now U.S. Pat. No.8,703,130, which is a divisional of U.S. application Ser. No.12/572,289, filed Oct. 2, 2009, now U.S. Pat. No. 8,268,314, whichclaims the benefit of European Patent Application No. 08017607.6, filedOct. 8, 2008, and European Patent Application No. 08021834.0, filed Dec.16, 2008, which are hereby incorporated by reference in their entirety.

SEQUENCE LISTING

This application contains a Sequence Listing which has been submittedvia EFS-Web and is hereby incorporated by reference in its entirety.Said ASCII copy, created on Mar. 11, 2020, is named Sequence_Listing.txtand is 207,671 bytes in size.

BACKGROUND OF THE INVENTION

The present invention relates to bispecific antibodies against humanvascular endothelial growth factor (VEGF/VEGF-A) and humanangiopoietin-2 (ANG-2), methods for their production, pharmaceuticalcompositions containing said antibodies, and uses thereof.

Angiogenesis is implicated in the pathogenesis of a variety of disorderswhich include solid tumors, intraocular neovascular syndromes such asproliferative retinopathies or age-related macular degeneration (AMD),rheumatoid arthritis, and psoriasis (Folkman, J., et al., J. Biol. Chem.267 (1992) 10931-10934; Klagsbrun, M., et al., Annu. Rev. Physiol. 53(1991) 217-239; and Garner, A., Vascular diseases, in: Pathobiology ofocular disease, A dynamic approach, Garner, A., and Klintworth, G. K.(eds.), 2nd edition, Marcel Dekker, New York (1994), pp 1625-1710). Inthe case of solid tumors, the neovascularization allows the tumor cellsto acquire a growth advantage and proliferative autonomy compared to thenormal cells. Accordingly, a correlation has been observed betweendensity of microvessels in tumor sections and patient survival in breastcancer as well as in several other tumors (Weidner, N., et al., N Engl JMed. 324 (1991) 1-8; Horak, E. R., et al., Lancet 340 (1992) 1120-1124;and Macchiarini, P., et al., Lancet 340 (1992) 145-146).

VEGF and Anti-VEGF Antibodies

Human vascular endothelial growth factor (VEGF/VEGF-A) (SEQ ID No: 105)is described in e.g. Leung, D. W., et al., Science 246 (1989) 1306-9;Keck, P. J., et al., Science 246 (1989) 1309-12 and Connolly, D. T., etal., J. Biol. Chem. 264 (1989) 20017-24. VEGF is involved in theregulation of normal and abnormal angiogenesis and neovascularizationassociated with tumors and intraocular disorders (Ferrara, N., et al.,Endocr. Rev. 18 (1997) 4-25; Berkman, R. A., et al., J. Clin. Invest. 91(1993) 153-159; Brown, L. F., et al., Human Pathol. 26 (1995) 86-91;Brown, L. F., et al., Cancer Res. 53 (1993) 4727-4735; Mattern, J., etal., Brit. J. Cancer. 73 (1996) 931-934; and Dvorak, H., et al., Am. J.Pathol. 146 (1995) 1029-1039). VEGF is a homodimeric glycoprotein thathas been isolated from several sources. VEGF shows highly specificmitogenic activity for endothelial cells. VEGF has important regulatoryfunctions in the formation of new blood vessels during embryonicvasculogenesis and in angiogenesis during adult life (Carmeliet, P., etal., Nature, 380 (1996) 435-439; Ferrara, N., et al., Nature, 380 (1996)439-442; reviewed in Ferrara and Davis-Smyth, Endocrine Rev., 18 (1997)4-25. The significance of the role played by VEGF has been demonstratedin studies showing that inactivation of a single VEGF allele results inembryonic lethality due to failed development of the vasculature(Carmeliet, P., et al., Nature, 380 (1996) 435-439; Ferrara, N., et al.,Nature, 380 (1996) 439-442. In addition VEGF has strong chemoattractantactivity towards monocytes, can induce the plasminogen activator and theplasminogen activator inhibitor in endothelial cells, and can alsoinduce microvascular permeability. Because of the latter activity, it issometimes referred to as vascular permeability factor (VPF). Theisolation and properties of VEGF have been reviewed; see Ferrara, N., etal., J. Cellular Biochem., 47 (1991) 211-218 and Connolly, J. CellularBiochem., 47 (1991) 219-223. Alternative mRNA splicing of a single VEGFgene gives rise to five isoforms of VEGF.

Anti-VEGF neutralizing antibodies suppress the growth of a variety ofhuman tumor cell lines in mice (Kim, I., et al., Nature 362 (1993)841-844; Warren, S. R., et al., J. Clin. Invest. 95 (1995) 1789-1797;Borgstrom, P., et al., Cancer Res. 56 (1996) 4032-4039; and Melnyk, O.,et al., Cancer Res. 56 (1996) 921-924). WO 94/10202, WO 98/45332, WO2005/00900 and WO 00/35956 refer to antibodies against VEGF. Humanizedmonoclonal antibody bevacizumab (sold under the trade name Avastin®) isan anti-VEGF antibody used in tumor therapy WO 98/45331).

Ranibizumab (trade name Lucentis®) is a monoclonal antibody fragmentderived from the same parent murine antibody as bevacizumab. It is muchsmaller than the parent molecule and has been affinity matured toprovide stronger binding to VEGF-A (WO 98/45331). It is ananti-angiogenic that has been approved to treat the “wet” type ofage-related macular degeneration (ARMD), a common form of age-relatedvision loss. Another anti-VEGF antibody is e.g. HuMab G6-31 describede.g. in US 2007/0141065.

ANG-2 and Anti-ANG-2 Antibodies

Human angiopoietin-2 (ANG-2) (alternatively abbreviated with ANGPT2 orANG2) (SEQ ID No: 106) is described in Maisonpierre, P. C., et al,Science 277 (1997) 55-60 and Cheung, A. H., et al., Genomics 48 (1998)389-91. The angiopoietins-1 and -2 (ANG-1 (SEQ ID No: 107) and ANG-2(SEQ ID No: 106)) were discovered as ligands for Ties, a family oftyrosine kinases that is selectively expressed within the vascularendothelium. Yancopoulos, G. D., et al., Nature 407 (2000) 242-48. Thereare now four definitive members of the angiopoietin family.Angiopoietin-3 and -4 (ANG-3 and ANG-4) may represent widely divergedcounterparts of the same gene locus in mouse and man. Kim, I., et al.,FEBS Let, 443 (1999) 353-56; Kim, I., et al., J Biol Chem 274 (1999)26523-28. ANG-1 and ANG-2 were originally identified in tissue cultureexperiments as agonist and antagonist, respectively (see for ANG-1:Davis, S., et al., Cell 87 (1996) 1161-69; and for ANG-2: Maisonpierre,P. C., et al., Science 277 (1997) 55-60). All of the known angiopoietinsbind primarily to Tie2, and both ANG-1 and -2 bind to Tie2 with anaffinity of 3 nM (K_(D)). Maisonpierre, P. C., et al., Science 277(1997) 55-60. ANG-1 was shown to support EC survival and to promoteendothelium integrity, Davis, S., et al., Cell 87 (1996) 1161-69; Kwak,H. J., et al., FEBS Lett 448 (1999) 249-53; Suri, C., et al., Science282 (1998) 468-71; Thurston, G., et al., Science 286 (1999) 251 1-14;Thurston, G., et al., Nat. Med. 6 (2000) 460-63, whereas ANG-2 had theopposite effect and promoted blood vessel destabilization and regressionin the absence of the survival factors VEGF or basic fibroblast growthfactor. Maisonpierre, P. C., et al., Science 277 (1997) 55-60. However,many studies of ANG-2 function have suggested a more complex situation.ANG-2 might be a complex regulator of vascular remodeling that plays arole in both vessel sprouting and vessel regression. Supporting suchroles for ANG-2, expression analysis reveals that ANG-2 is rapidlyinduced, together with VEGF, in adult settings of angiogenic sprouting,whereas ANG-2 is induced in the absence of VEGF in settings of vascularregression. Holash, J., et al., Science 284 (1999) 1994-98; Holash, J.,et al., Oncogene 18 (1999) 5356-62. Consistent with a context-dependentrole, ANG-2 specifically binds to the same endothelial-specificreceptor, Tie-2, which is activated by ANG-1, but has context-dependenteffects on its activation. Maisonpierre, P. C., et al., Science 277(1997) 55-60.

Corneal angiogenesis assays have shown that both ANG-1 and ANG-2 hadsimilar effects, acting synergistically with VEGF to promote growth ofnew blood vessels. Asahara, T., et al., Circ. Res. 83 (1998) 233-40. Thepossibility that there was a dose-dependent endothelial response wasraised by the observation that, in vitro at high concentration, ANG-2can also be pro-angiogenic. Kim, I., et al., Oncogene 19 (2000) 4549-52.At high concentration, ANG-2 acts as an apoptosis survival factor forendothelial cells during serum deprivation apoptosis through activationof Tie2 via P1-3 Kinase and Akt pathway. Kim, I., et al., Oncogene 19(2000) 4549-52.

Other in vitro experiments suggested that, during sustained exposure,the effects of ANG-2 may progressively shift from that of an antagonistto an agonist of Tie2, and, at later time points, it may contributedirectly to vascular tube formation and neovessel stabilization.Teichert-Kuliszewska, K., et al., Cardiovasc. Res. 49 (2001) 659-70.Furthermore, if ECs were cultivated on fibrin gel, activation of Tie2with ANG-2 was also observed, perhaps suggesting that the action ofANG-2 could depend on EC differentiation state. Teichert-Kuliszewska,K., et al., Cardiovasc. Res. 49 (2001) 659-70. In microvascular ECcultured in a three-dimensional collagen gel, ANG-2 can also induce Tie2activation and promote formation of capillary-like structures.Mochizuki, Y., et al., J. Cell. Sci. 115 (2002) 175-83. Use of a 3-Dspheroidal coculture as an in-vitro model of vessel maturationdemonstrated that direct contact between ECs and mesenchymal cellsabrogates responsiveness to VEGF, whereas the presence of VEGF and ANG-2induced sprouting. Korff, T., et al., Faseb J. 15 (2001) 447-57. Etoh,T. H. et al. demonstrated that ECs that constitutively express Tie2, theexpression of MMP-1, -9 and u-PA were strongly upregulated by ANG-2 inthe presence of VEGF. Etoh, T., et al., Cancer Res. 61 (2001) 2145-53.With an in vivo pupillary membrane model, Lobov, I. B. et al. showedthat ANG-2 in the presence of endogenous VEGF promotes a rapid increasein capillary diameter, remodeling of the basal lamina, proliferation andmigration of endothelial cells, and stimulates sprouting of new bloodvessels. Lobov, I. B., et al., Proc. Natl. Acad. Sci. USA 99 (2002)11205-10. By contrast, ANG-2 promotes endothelial cell death and vesselregression without endogenous VEGF. Lobov, I. B., et al., Proc. Natl.Acad. Sci. USA 99 (2002) 11205-10. Similarly, with an in vivo tumormodel, Vajkoczy, P., et al. demonstrated that multicellular aggregatesinitiate vascular growth by angiogenic sprouting via the simultaneousexpression of VEGFR-2 and ANG-2 by host and tumor endothelium. Vajkoczy,P., et al., J. Clin. Invest. 109 (2002) 777-85. This model illustratedthat the established microvasculature of growing tumors is characterizedby a continuous remodeling, putatively mediated by the expression ofVEGF and ANG-2. Vajkoczy, P., et al., J Clin. Invest. 09 (2002) 777-85.

Knock-out mouse studies of Tie-2 and Angiopoietin-1 show similarphenotypes and suggest that Angiopoietin-1 stimulated Tie-2phosphorylation mediates remodeling and stabilization of developingvessel, promoting blood vessel maturation during angiogenesis andmaintenance of endothelial cell-support cell adhesion (Dumont, J., etal., Genes & Development, 8 (1994) 1897-1909; Sato, T. N., Nature, 376(1995) 70-74; (Thurston, G., et al., Nature Medicine: 6 (2000) 460-463).The role of Angiopoietin-1 is thought to be conserved in the adult,where it is expressed widely and constitutively (Hanahan, D., Science,277 (1997) 48-50; Zagzag, D., et al., Exp Neurology, 159:391-400(1999)). In contrast, Angiopoietin-2 expression is primarily limited tosites of vascular remodeling where it is thought to block theconstitutive stabilizing or maturing function of Angiopoietin-1,allowing vessels to revert to, and remain in, a plastic state which maybe more responsive to sprouting signals (Hanahan, D., 1997; Holash, J.,et al., Orzcogerze 18 (199) 5356-62; Maisonpierre, P. C., 1997). Studiesof Angiopoietin-2 expression in pathological angiogenesis have foundmany tumor types to show vascular Angiopoietin-2 expression(Maisonpierre, P. C., et al., Science 277 (1997) 55-60). Functionalstudies suggest Angiopoietin-2 is involved in tumor angiogenesis andassociate Angiopoietin-2 overexpression with increased tumor growth in amouse xenograft model (Ahmad, S. A., et al., Cancer Res., 61 (2001)1255-1259). Other studies have associated Angiopoietin-2 overexpressionwith tumor hypervascularity (Etoh, T., et al., Cancer Res. 61 (2001)2145-53; Tanaka, F., et al., Cancer Res. 62 (2002) 124-29).

In recent years Angiopoietin-1, Angiopoietin-2 and/or Tie-2 have beenproposed as possible anti-cancer therapeutic targets. For example U.S.Pat. Nos. 6,166,185, 5,650,490 and 5,814,464 each disclose anti-Tie-2ligand and receptor antibodies. Studies using soluble Tie-2 werereported to decrease the number and size of tumors in rodents (Lin,1997; Lin 1998). Siemester, G., et al. Siemeister, G., et al., CancerRes. 59 (1999) 3185-91 generated human melanoma cell lines expressingthe extracellular domain of Tie-2, injected these into nude mice andreported soluble Tie-2 to result in significant inhibition of tumorgrowth and tumor angiogenesis. Given that both Angiopoietin-1 andAngiopoietin-2 bind to Tie-2, it is unclear from these studies whetherAngiopoietin-1, Angiopoietin-2 or Tie-2 would be an attractive targetfor anti-cancer therapy. However, effective anti-Angiopoietin-2 therapyis thought to be of benefit in treating diseases such as cancer, inwhich progression is dependant on aberrant angiogenesis where blockingthe process can lead to prevention of disease advancement (Follunan, J.,Nature Medicine. 1 (1995) 27-31).

In addition some groups have reported the use of antibodies and peptidesthat bind to Angiopoietin-2. See, for example, U.S. Pat. No. 6,166,185and US 2003/10124129. WO 03/030833, WO 2006/068953, WO 03/057134 or US2006/0122370.

Study of the effect of focal expression of Angiopoietin-2 has shown thatantagonizing the Angiopoietin-1/Tie-2 signal loosens the tight vascularstructure thereby exposing ECs to activating signals from angiogenesisinducers, e.g. VEGF (Hanahan, D., Science, 277 (1997) 48-50). Thispro-angiogenic effect resulting from inhibition of Angiopoietin-1indicates that anti-Angiopoietin-1 therapy would not be an effectiveanti-cancer treatment.

ANG-2 is expressed during development at sites where blood vesselremodeling is occurring. Maisonpierre, P. C., et al., Science 277 (1997)55-60. In adult individuals, ANG-2 expression is restricted to sites ofvascular remodeling as well as in highly vascularized tumors, includingglioma, Osada, H., et al., Int. J. Oncol. 18 (2001) 305-09); Koga, K.,et al., Cancer Res. 61 (2001) 6248-54, hepatocellular carcinoma, Tanaka,S., et al., J. Clin. Invest. 103 (1999) 341-45, gastric carcinoma, Etoh,T., et al., Cancer Res. 61 (2001) 2145-53; Lee, J. H., et al., Int. J.Oncol. 18 (2001) 355-61, thyroid tumor, Bunone, G., et al., Am J Pathol155 (1999) 1967-76 non-small cell lung cancer, Wong, M. P., et al., LungCancer 29 (2000) 11-22, and cancer of colon, Ahmad, S. A., et al.,Cancer 92 (2001) 1138-43, and prostate Wurmbach, J. H., et al.,Anticancer Res. 20 (2000) 5217-20. Some tumor cells are found to expressANG-2. For example, Tanaka, S., et al., J. Clin. Invest. 103 (1999)341-45 detected ANG-2 mRNA in 10 out of 12 specimens of humanhepatocellular carcinoma (HCC). Ellis' group reported that ANG-2 isexpressed ubiquitously in tumor epithelium. Ahmad, S. A., et al., Cancer92 (2001) 1138-43. Other investigators reported similar findings. Chen,L., et al., J. Tongji Med. Univ. 21 (2001) 228-35. By detecting ANG-2mRNA levels in archived human breast cancer specimens, Sfiligoi, C., etal., Int. J. Cancer 103 (2003) 466-74 reported that ANG-2 mRNA issignificantly associated with auxiliary lymph node invasion, shortdisease-free time and poor overall survival. Tanaka, F., et al., CancerRes. 62 (2002) 7124-29 reviewed a total of 236 patients of non-smallcell lung cancer (NSCLC) with pathological stage-I to -IIIA,respectively. Using immunohistochemistry, they found that 16.9% of theNSCLC patients were ANG-2 positive. The microvessel density for ANG-2positive tumor is significantly higher than that of ANG-2 negative. Suchan angiogenic effect of ANG-2 was seen only when VEGF expression washigh. Moreover, positive expression of ANG-2 was a significant factor topredict a poor postoperative survival. Tanaka, F., et al., Cancer Res.62 (2002) 7124-29. However, they found no significant correlationbetween Ang-1 expression and the microvessel density. Tanaka, F., etal., Cancer Res. 62 (2002) 7124-29. These results suggest that ANG-2 isan indicator of poor prognosis patients with several types of cancer.

Recently, using an ANG-2 knockout mouse model, Yancopoulos' groupreported that ANG-2 is required for postnatal angiogenesis. Gale, N. W.,et al., Dev. Cell 3 (2002) 411-23. They showed that the developmentallyprogrammed regression of the hyaloid vasculature in the eye does notoccur in the ANG-2 knockout mice and their retinal blood vessels fail tosprout out from the central retinal artery. Gale, N. W., et al., Dev.Cell 3 (2002) 411-23. They also found that deletion of ANG-2 results inprofound defects in the patterning and function of the lymphaticvasculature. Gale, N. W., et al., Dev. Cell 3 (200) 411-23. Geneticrescue with Ang-1 corrects the lymphatic, but not the angiogenesisdefects. Gale, N. W., et al., Dev. Cell 3 (2002) 411-23.

Peters and his colleagues reported that soluble Tie2, when deliveredeither as recombinant protein or in a viral expression vector, inhibitedin vivo growth of murine mammary carcinoma and melanoma in mouse models.Lin, P., et al., Proc. Natl. Acad. Sci. USA 95 (1998) 8829-34; Lin, P.,et al., J. Clin. Invest. 100 (1997) 2072-78. Vascular densities in thetumor tissues so treated were greatly reduced. In addition, soluble Tie2blocked angiogenesis in the rat corneal stimulated by tumor cellconditioned media. Lin, P., et al., J. Clin. Invest. 100 (1997) 2072-78.Furthermore, Isner and his team demonstrated that addition of ANG-2 toVEGF promoted significantly longer and more circumferentialneovascularity than VEGF alone. Asahara, T., et al., Circ. Res. 83(1998) 233-40. Excess soluble Tie2 receptor precluded modulation ofVEGF-induced neovascularization by ANG-2. Asahara, T., et al., Circ.Res. 83 (1998) 233-40. Siemeister, G., et al., Cancer Res. 59 (1999)3185-91 showed with nude mouse xenografts that overexpression of theextracellular ligand-binding domains of either Flt-1 or Tie2 in thexenografts results in significant inhibition of pathway could not becompensated by the other one, suggesting that the VEGF receptor pathwayand the Tie2 pathway should be considered as two independent mediatorsessential for the process of in vivo angiogenesis. Siemeister, G., etal., Cancer Res. 59:3 (1999) 3185-91. This is proven by a more recentpublication by White, R., R., et al., Proc. Natl. Acad. Sci. USA 100(2003) 5028-33. In their study, it was demonstrated that anuclease-resistant RNA aptamer that specifically binds and inhibitsANG-2 significantly inhibited neovascularization induced by bFGF in therat corneal micropocket angiogenesis model.

Bispecific Antibodies

A wide variety of recombinant antibody formats have been developed inthe recent past, e.g. tetravalent bispecific antibodies by fusion of,e.g., an IgG antibody format and single chain domains (see e.g. Coloma,M. J., et al., Nature Biotech 15 (1997) 159-163; WO 2001/077342; andMorrison, S. L., Nature Biotech 25 (2007) 1233-1234).

Also several other new formats wherein the antibody core structure (IgA,IgD, IgE, IgG or IgM) is no longer retained such as dia-, tria- ortetrabodies, minibodies, several single chain formats (scFv, Bis-scFv),which are capable of binding two or more antigens, have been developed(Holliger, P., et al., Nature Biotech 23 (2005) 1126-1136; Fischer, N.,Léger, O., Pathobiology 74 (2007) 3-14; Shen, J., et al., Journal ofImmunological Methods 318 (2007) 65-74; Wu, C., et al., Nature Biotech.25 (2007) 1290-1297).

All such formats use linkers either to fuse the antibody core (IgA, IgD,IgE, IgG or IgM) to a further binding protein (e.g. scFv) or to fusee.g. two Fab fragments or scFvs (Fischer, N., Léger, O., Pathobiology 74(2007) 3-14). It has to be kept in mind that one may want to retaineffector functions, such as e.g. complement-dependent cytotoxicity (CDC)or antibody dependent cellular cytotoxicity (ADCC), which are mediatedthrough the Fc receptor binding, by maintaining a high degree ofsimilarity to naturally occurring antibodies.

In WO 2007/024715 are reported dual variable domain immunoglobulins asengineered multivalent and multispecific binding proteins. A process forthe preparation of biologically active antibody dimers is reported inU.S. Pat. No. 6,897,044. Multivalent Fv antibody construct having atleast four variable domains which are linked with each over via peptidelinkers are reported in U.S. Pat. No. 7,129,330. Dimeric and multimericantigen binding structures are reported in US 2005/0079170. Tri- ortetra-valent monospecific antigen-binding protein comprising three orfour Fab fragments bound to each other covalently by a connectingstructure, which protein is not a natural immunoglobulin are reported inU.S. Pat. No. 6,511,663. In WO 2006/020258 tetravalent bispecificantibodies are reported that can be efficiently expressed in prokaryoticand eukaryotic cells, and are useful in therapeutic and diagnosticmethods. A method of separating or preferentially synthesizing dimerswhich are linked via at least one interchain disulfide linkage fromdimers which are not linked via at least one interchain disulfidelinkage from a mixture comprising the two types of polypeptide dimers isreported in US 2005/0163782. Bispecific tetravalent receptors arereported in U.S. Pat. No. 5,959,083. Engineered antibodies with three ormore functional antigen binding sites are reported in WO 2001/077342.

Multispecific and multivalent antigen-binding polypeptides are reportedin WO 1997/001580. WO 1992/004053 reports homoconjugates, typicallyprepared from monoclonal antibodies of the IgG class which bind to thesame antigenic determinant are covalently linked by syntheticcross-linking. Oligomeric monoclonal antibodies with high avidity forantigen are reported in WO 1991/06305 whereby the oligomers, typicallyof the IgG class, are secreted having two or more immunoglobulinmonomers associated together to form tetravalent or hexavalent IgGmolecules. Sheep-derived antibodies and engineered antibody constructsare reported in U.S. Pat. No. 6,350,860, which can be used to treatdiseases wherein interferon gamma activity is pathogenic. In US2005/0100543 are reported targetable constructs that are multivalentcarriers of bi-specific antibodies, i.e., each molecule of a targetableconstruct can serve as a carrier of two or more bi-specific antibodies.Genetically engineered bispecific tetravalent antibodies are reported inWO 1995/009917. In WO 2007/109254 stabilized binding molecules thatconsist of or comprise a stabilized scFv are reported.

Combination of VEGF and ANG-2 Inhibitors

WO 2007/068895 refers to a combination of an ANG-2 antagonist and aVEGF, KDR and/or FLTL antagonists. WO 2007/089445 refers to ANG-2 andVEGF inhibitor combinations.

WO 2003/106501 refers to fusion proteins binding to Angiopoetin andcontaining a multimerization domain. WO 2008/132568 fusion proteinsbinding to Angiopoetin and VEGF.

SUMMARY OF THE INVENTION

A first aspect of the current invention is a bispecific antibody thatbinds specifically to human vascular endothelial growth factor (VEGF)and human angiopoietin-2 (ANG-2), said antibody comprising a firstantigen-binding site that specifically binds to human VEGF and a secondantigen-binding site that specifically binds to human ANG-2.

Said bispecific antibodies are at least bivalent and may be trivalent,tetravalent or multivalent. Preferably the bispecific antibody accordingto the invention is bivalent, trivalent or tetravalent.

A further aspect of the invention is a nucleic acid molecule encodingsaid bispecific antibody.

The invention further provides an expression vector which comprises saidnucleic acid according to the invention and which is capable ofexpressing said nucleic acid in a prokaryotic or eukaryotic host cell.

Also provided is are host cells containing such vectors for therecombinant production of an antibody according to the invention.

The invention further comprises a prokaryotic or eukaryotic host cellcomprising a vector according to the invention.

The invention further comprises a method for the production of abispecific antibody according to the invention, comprising expressing anucleic acid according to the invention in a prokaryotic or eukaryotichost cell and recovering said bispecific antibody from said cell or thecell culture supernatant. The invention further comprises the antibodyobtained by such a recombinant method.

Still further aspects of the invention are a pharmaceutical compositioncomprising said bispecific antibody, said composition for the treatmentof cancer, the use of said bispecific antibody for the manufacture of amedicament for the treatment of cancer, a method of treatment of patientsuffering from cancer by administering said bispecific antibody. to apatient in the need of such treatment.

The bispecific antibodies according to the invention show benefits forhuman patients in need of a VEGF and ANG-2 targeting therapy. Theantibodies according to the invention have new and inventive propertiescausing a benefit for a patient suffering from such a disease,especially suffering from cancer. Surprisingly it has found out that thebispecific antibodies according to the invention are more effective intumor growth and/or inhibition of tumor angiogenesis compared tocombination of the respective monospecific parent antibodies.

Description of the Amino acid Sequences SEQ ID NO: 1 heavy chain CDR3,<VEGF>bevacizumab SEQ ID NO: 2 heavy chain CDR2, <VEGF>bevacizumab SEQID NO: 3 heavy chain CDR1, <VEGF>bevacizumab SEQ ID NO: 4 light chainCDR3, <VEGF>bevacizumab SEQ ID NO: 5 light chain CDR2, <VEGF>bevacizumabSEQ ID NO: 6 light chain CDR1, <VEGF>bevacizumab SEQ ID NO: 7 heavychain variable domain, <VEGF>bevacizumab SEQ ID NO: 8 light chainvariable domain, <VEGF>bevacizumab SEQ ID NO: 9 heavy chain CDR3,<VEGF>ranibizumab SEQ ID NO: 10 heavy chain CDR2, <VEGF>ranibizumab SEQID NO: 11 heavy chain CDR1, <VEGF>ranibizumab SEQ ID NO: 12 light chainCDR3, <VEGF>ranibizumab SEQ ID NO: 13 light chain CDR2,<VEGF>ranibizumab SEQ ID NO: 14 light chain CDR1, <VEGF>ranibizumab SEQID NO: 15 heavy chain variable domain, <VEGF>ranibizumab SEQ ID NO: 16light chain variable domain, <VEGF>ranibizumab SEQ ID NO: 17 heavy chainCDR3, <VEGF>HuMab G6-31 SEQ ID NO: 18 heavy chain CDR2, <VEGF> HuMabG6-31 SEQ ID NO: 19 heavy chain CDR1, <VEGF> HuMab G6-31 SEQ ID NO: 20light chain CDR3, <VEGF> HuMab G6-31 SEQ ID NO: 21 light chain CDR2,<VEGF> HuMab G6-31 SEQ ID NO: 22 light chain CDR1, <VEGF> HuMab G6-31SEQ ID NO: 23 heavy chain variable domain, <VEGF> HuMab G6- 31 SEQ IDNO: 24 light chain variable domain, <VEGF> HuMab G6- 31 SEQ ID NO: 25heavy chain CDR3, <ANG-2> Mab 536 SEQ ID NO: 26 heavy chain CDR2,<ANG-2> Mab 536 SEQ ID NO: 27 heavy chain CDR1, <ANG-2> Mab 536 SEQ IDNO: 28 light chain CDR3, <ANG-2> Mab 536 SEQ ID NO: 29 light chain CDR2,<ANG-2> Mab 536 SEQ ID NO: 30 light chain CDR1, <ANG-2> Mab 536 SEQ IDNO: 31 heavy chain variable domain, <ANG-2> Mab 536 SEQ ID NO: 32 lightchain variable domain, <ANG-2> Mab 536 SEQ ID NO: 33 (G4S)4 linker SEQID NO: 34 lambda light chain constant region SEQ ID NO: 35 human heavychain constant region derived from IgG1 SEQ ID NO: 36 human heavy chainconstant region derived from IgG4 SEQ ID NO: 37 kappa light chainconstant region SEQ ID NO: 38 heavy chain CDR3, <ANG-2> Ang2s_R3_LC03SEQ ID NO: 39 heavy chain CDR2, <ANG-2> Ang2s_R3_LC03 SEQ ID NO: 40heavy chain CDR1, <ANG-2> Ang2s_R3_LC03 SEQ ID NO: 41 light chain CDR3,<ANG-2> Ang2s_R3_LC03 SEQ ID NO: 42 light chain CDR2, <ANG-2>Ang2s_R3_LC03 SEQ ID NO: 43 light chain CDR1, <ANG-2> Ang2s_R3_LC03 SEQID NO: 44 heavy chain variable domain, <ANG-2> Ang2s_R3_LC03 SEQ ID NO:45 light chain variable domain, <ANG-2> Ang2s_R3_LC03 SEQ ID NO: 46heavy chain CDR3, <ANG-2>Ang2i_LC06 SEQ ID NO: 47 heavy chain CDR2,<ANG-2> Ang2i_LC06 SEQ ID NO: 48 heavy chain CDR1, <ANG-2>Ang2i_LC06 SEQID NO: 49 light chain CDR3, <ANG-2>Ang2i_LC06 SEQ ID NO: 50 light chainCDR2, <ANG-2>Ang2i_LC06 SEQ ID NO: 51 light chain CDR1,<ANG-2>Ang2i_LC06 SEQ ID NO: 52 heavy chain variable domain,<ANG-2>Ang2i_LC06 SEQ ID NO: 53 light chain variable domain,<ANG-2>Ang2i_LC06 SEQ ID NO: 54 heavy chain CDR3, <ANG-2>Ang2i_LC07 SEQID NO: 55 heavy chain CDR2, <ANG-2>Ang2i_LC07 SEQ ID NO: 56 heavy chainCDR1, <ANG-2>Ang2i_LC07 SEQ ID NO: 57 light chain CDR3,<ANG-2>Ang2i_LC07 SEQ ID NO: 58 light chain CDR2, <ANG-2>Ang2i_LC07 SEQID NO: 59 light chain CDR1, <ANG-2>Ang2i_LC07 SEQ ID NO: 60 heavy chainvariable domain, <ANG-2>Ang2i_LC07 SEQ ID NO: 61 light chain variabledomain, <ANG-2>Ang2i_LC07 SEQ ID NO: 62 heavy chain CDR3,<ANG-2>Ang2k_LC08 SEQ ID NO: 63 heavy chain CDR2, <ANG-2> Ang2k_LC08 SEQID NO: 64 heavy chain CDR1, <ANG-2> Ang2k_LC08 SEQ ID NO: 65 light chainCDR3, <ANG-2> Ang2k_LC08 SEQ ID NO: 66 light chain CDR2, <ANG-2>Ang2k_LC08 SEQ ID NO: 67 light chain CDR1, <ANG-2> Ang2k_LC08 SEQ ID NO:68 heavy chain variable domain, <ANG-2> Ang2k_LC08 SEQ ID NO: 69 lightchain variable domain, <ANG-2> Ang2k_LC08 SEQ ID NO: 70 heavy chainCDR3, <ANG-2> Ang2s_LC09 SEQ ID NO: 71 heavy chain CDR2, <ANG-2>Ang2s_LC09 SEQ ID NO: 72 heavy chain CDR1, <ANG-2> Ang2s_LC09 SEQ ID NO:73 light chain CDR3, <ANG-2> Ang2s_LC09 SEQ ID NO: 74 light chain CDR2,<ANG-2> Ang2s_LC09 SEQ ID NO: 75 light chain CDR1, <ANG-2> Ang2s_LC09SEQ ID NO: 76 heavy chain variable domain, <ANG-2> Ang2s_LC09 SEQ ID NO:77 light chain variable domain, <ANG-2> Ang2s_LC09 SEQ ID NO: 78 heavychain CDR3, <ANG-2> Ang2i_LC10 SEQ ID NO: 79 heavy chain CDR2, <ANG-2>Ang2i_LC10 SEQ ID NO: 80 heavy chain CDR1, <ANG-2> Ang2i_LC10 SEQ ID NO:81 light chain CDR3, <ANG-2> Ang2i_LC10 SEQ ID NO: 82 light chain CDR2,<ANG-2> Ang2i_LC10 SEQ ID NO: 83 light chain CDR1, <ANG-2> Ang2i_LC10SEQ ID NO: 84 heavy chain variable domain, <ANG-2> Ang2i_LC10 SEQ ID NO:85 light chain variable domain, <ANG-2> Ang2i_LC10 SEQ ID NO: 86 heavychain CDR3, <ANG-2> Ang2k_LC11 SEQ ID NO: 87 heavy chain CDR2, <ANG-2>Ang2k_LC11 SEQ ID NO: 88 heavy chain CDR1, <ANG-2> Ang2k_LC11 SEQ ID NO:89 light chain CDR3, <ANG-2> Ang2k_LC11 SEQ ID NO: 90 light chain CDR2,<ANG-2> Ang2k_LC11 SEQ ID NO: 91 light chain CDR1, <ANG-2> Ang2k_LC11SEQ ID NO: 92 heavy chain variable domain, <ANG-2> Ang2k_LC11 SEQ ID NO:93 light chain variable domain, <ANG-2> Ang2k_LC11 SEQ ID NO: 94 heavychain CDR3, <VEGF>B20-4.1 SEQ ID NO: 95 heavy chain CDR2, <VEGF>B20-4.1SEQ ID NO: 96 heavy chain CDR1, <VEGF>B20-4.1 SEQ ID NO: 97 light chainCDR3, <VEGF>B20-4.1 SEQ ID NO: 98 light chain CDR2, <VEGF>B20-4.1 SEQ IDNO: 99 light chain CDR1, <VEGF>B20-4.1 SEQ ID NO: 100 heavy chainvariable domain, <VEGF>B20-4.1 SEQ ID NO: 101 light chain variabledomain, <VEGF>B20-4.1 SEQ ID NO: 102 bevacizumab heavy chain Ang2i_LC06scFv fusion peptide of <VEGF-ANG-2> TvAb-2441-bevacizumab-LC06 SEQ IDNO: 103 bevacizumab heavy chain Ang2i_LC08 scFv fusion peptide of<VEGF-ANG-2> TvAb-2441-bevacizumab-LC08 SEQ ID NO: 104 light chain ofbevacizumab SEQ ID NO: 105 Human vascular endothelial growth factor(VEGF) SEQ ID NO: 106 Human angiopoietin-2 (ANG-2) SEQ ID NO: 107 Humanangiopoietin-1 (ANG-1) SEQ ID NO: 108 Human Tie-2 receptor SEQ ID NO:109 Heavy chain 1 of bispecific, tetravalent single chain Fab<VEGF-ANG-2> antibody molecule scFAb-Bevacizumab-LC06-2620 SEQ ID NO:110 Light chain of bispecific, tetravalent single chain Fab <VEGF-ANG-2>antibody molecule scFAb-Bevacizumab-LC06-2620 SEQ ID NO: 111 Heavy chain1 of bispecific, tetravalent single chain Fab <VEGF-ANG-2> antibodymolecule scFab-Bevacizumab-Ang2i-LC06-2640 SEQ ID NO: 112 Light chain ofbispecific, tetravalent single chain Fab <VEGF-ANG-2> antibody moleculescFab-Bevacizumab-Ang2i-LC06-2640 SEQ ID NO: 113 Heavy chain 1 ofbispecific, tetravalent single chain Fab <VEGF-ANG-2> antibody moleculescFab-Bevacizumab-Ang2i-LC06-2641 SEQ ID NO: 114 Light chain ofbispecific, tetravalent single chain Fab <VEGF-ANG-2> antibody moleculescFab-Bevacizumab-Ang2i-LC06-2641 SEQ ID NO: 115 Heavy chain 1 ofbispecific, trivalent single chain Fab <VEGF-ANG-2> antibody moleculeBevacizumab-LC06-KiH-C-scFab SEQ ID NO: 116 Heavy chain 2 of bispecific,trivalent single chain Fab <VEGF-ANG-2> antibody moleculeBevacizumab-LC06-KiH-C-scFab SEQ ID NO: 117 Light chain of bispecific,trivalent single chain Fab <VEGF-ANG-2> antibody moleculeBevacizumab-LC06-KiH-C-scFab SEQ ID NO: 118 Heavy chain 1 of bispecific,trivalent <VEGF-ANG-2> antibody molecule Bevacizumab-LC06-C-Fab-6CSS SEQID NO: 119 Heavy chain 2 of bispecific, trivalent <VEGF-ANG-2> antibodymolecule Bevacizumab-LC06-C-Fab-6CSS SEQ ID NO: 120 Light chain ofbispecific, trivalent <VEGF-ANG-2>antibody moleculeBevacizumab-LC06-C-Fab-6CSS SEQ ID NO: 121 Heavy chain 1 of bispecific,bivalent domain exchanged <VEGF-ANG-2> antibody moleculeBevacizumab-LC06-CH1-CL SEQ ID NO: 122 Heavy chain 2 of bispecific,bivalent domain exchanged <VEGF-ANG-2> antibody moleculeBevacizumab-LC06-CH1-CL SEQ ID NO: 123 Light chain 1 of bispecific,bivalent domain exchanged <VEGF-ANG-2> antibody moleculeBevacizumab-LC06-CH1-CL SEQ ID NO: 124 Light chain 2 of bispecific,bivalent domain exchanged <VEGF-ANG-2> antibody moleculeBevacizumab-LC06-CH1-CL SEQ ID NO: 125 Heavy chain 1 of bispecific,bivalent domain exchanged <VEGF-ANG-2> antibody moleculeBevacizumab-LC06-VH-VL SEQ ID NO: 126 Heavy chain 2 of bispecific,bivalent domain exchanged <VEGF-ANG-2> antibody moleculeBevacizumab-LC06-VH-VL SEQ ID NO: 127 Light chain 1 of bispecific,bivalent domain exchanged <VEGF-ANG-2> antibody moleculeBevacizumab-LC06- VH-VL SEQ ID NO: 128 Light chain 2 of bispecific,bivalent domain exchanged <VEGF-ANG-2> antibody moleculeBevacizumab-LC06- VH-VL SEQ ID NO: 129 Heavy chain 1 of bispecific,bivalent domain exchanged <VEGF-ANG-2> antibody moleculeBevacizumab-LC06-VH-VL-SS SEQ ID NO: 130 Heavy chain 2 of bispecific,bivalent domain exchanged <VEGF-ANG-2> antibody moleculeBevacizumab-LC06-VH-VL-SS SEQ ID NO: 131 Light chain 1 of bispecific,bivalent domain exchanged <VEGF-ANG-2> antibody moleculeBevacizumab-LC06- VH-VL-SS SEQ ID NO: 132 Light chain 2 of bispecific,bivalent domain exchanged <VEGF-ANG-2> antibody moleculeBevacizumab-LC06- VH-VL-SS SEQ ID NO: 133 Heavy chain 1 of bispecific,bivalent ScFab-Fc fusion <VEGF-ANG-2> antibody moleculeBevacizumab-LC06-N-scFab SEQ ID NO: 134 Heavy chain 2 of bispecific,bivalent ScFab-Fc fusion <VEGF-ANG-2> antibody moleculeBevacizumab-LC06-N-scFab SEQ ID NO: 135 Heavy chain 1 of bispecific,bivalent ScFab-Fc fusion <VEGF-ANG-2> antibody moleculeBevacizumab-LC06-N-scFabSS SEQ ID NO: 136 Heavy chain 2 of bispecific,bivalent ScFab-Fc fusion <VEGF-ANG-2> antibody moleculeBevacizumab-LC06-N-scFabSS

DESCRIPTION OF THE FIGURES

FIG. 1A Schematic structure of one tetravalent embodiment of abispecific antibody according to the invention which binds to VEGF andANG-2, wherein one of the Antigens A or B is VEGF, while the other isANG-2. The structure is based on a full length antibody binding toAntigen A, to which two (optionally disulfide-stabilized) single chainFv's binding to Antigen B, are linked via the a peptide-linker.

FIG. 1B Schematic representation of the generated bispecific tetravalentantibodies using the TvAb nomenclature (see Examples)—either without orwith disulfide stabilization of the scFv

FIG. 2A Schematic representation of disulfide-stabilized <VEGF-ANG-2>bispecific tetravalent antibody (=<VEGF-ANG-2> TvAb6; No. 2331, seeTable 3)

FIG. 2B Plasmid maps of the modified heavy chain and the light vectorsused for the expression of disulfide-stabilized <VEGF-ANG-2> TvAb6

FIG. 3 SDS-PAGE of purified disulfide-stabilized <VEGF-ANG-2> TvAb6 incomparison to the “standard” human IgG1 antibody G6-31 (<VEGF> HuMabG6-31) under reducing and non-reducing conditions

FIGS. 4A-C Size exclusion chromatography of purifieddisulfide-stabilized <VEGF-ANG-2> TvAb6 in comparison to the “standard”human IgG1 antibody G6-31 shows that disulfide-stabilized TvAb6 does notform again aggregates upon purification. FIG. 4A shows the Protein Apool; FIG. 4B shows the SEC pool, after purification; and FIG. 4C showsthe SEC pool after 24 hours at 4° C.

FIG. 5 Schematic view and results from VEGF binding ELISA.Disulfide-stabilized <VEGF-ANG-2> TvAb6 binds to VEGF comparably to<VEGF> G6-31. <ANG-2> Mab536 does not bind to VEGF

FIG. 6A Schematic view and results from ANG-2 binding ELISA.disulfide-stabilized <VEGF-ANG-2>. TvAb6 binds to ANG-2 comparably to<ANG-2> Mab536. <VEGF> G6-31 does not bind to ANG-2.

FIG. 6B Schematic view and results from ANG-2 binding analysis bysurface plasmon resonance (Biacore). Disulfide-stabilized <VEGF-ANG-2>TvAb6 binds to ANG-2 with comparable affinity as <ANG-2> Mab536.

FIG. 7 Schematic view and results from VEGF-ANG-2 bridging ELISA.

Disulfide-stabilized <VEGF-ANG-2> TvAb6 binds simultaneously to VEGF andANG-2 whereas <VEGF> G6-31 and <ANG-2> Mab536 are not capable of bindingsimultaneously to VEGF and ANG-2.

FIG. 8a Efficacy of disulfide-stabilized <VEGF-ANG-2> TvAb6 incomparison to <ANG-2> Mab536, <VEGF> G6-31 and the combination of Mab536and G6-31 in the staged subcutaneous Colo205 xenograft model in Scidbeige mice (study ANG2_Pz_Colo205_003)

FIG. 8b Efficacy of disulfide-stabilized <VEGF-ANG-2> TvAb6 incomparison to <ANG-2> Mab536, <VEGF> G6-31 and the combination of Mab536and G6-31 in the staged subcutaneous Colo205 xenograft model in Scidbeige mice (study ANG2_Pz_Colo205_005)

FIG. 9 Blocking of VEGF-induced tube formation by the bispecifictetravalent antibody <VEGF-ANG-2> TvAb6—Results

FIG. 10A+B Blocking of VEGF-induced tube formation by thedisulfide-stabilized <VEGF-ANG-2> TvAb6—Quantitative analysis

FIG. 11 Schematic view of VEGF binding analysis by surface plasmonresonance

(Biacore).

FIG. 12 Kinetic characteristics of the two <VEGF> antibodies<VEGF-Ang-2> TvAb6 and <VEGF> G6-31 in a K_(A)-K_(D) plot.

FIG. 13 Schematic view of surface plasmon resonance (Biacore) assay todetect simultaneous binding of ANGPT2 and VEGF to bispecific antibodies

FIG. 14 Results from surface plasmon resonance (Biacore) experimentsshowing that TvAb6 binds simultaneously to ANGPT2 and VEGF.

FIG. 15A+B A) Schematic representation of the bispecific andsimultaneous Biacore binding assay of the <VEGF-ANG-2> bispecificantibodies. B) Biacore data demonstrating simultaneous binding of ANG-2and VEGF to TvAb-2441-bevacicumab_LC06

FIG. 16A+B Tie2 phosphorylation of the bispecific antibodies<VEGF-ANG-2>TvAb-2441-bevacizumab-LC06 and <VEGF-ANG-2>TvAb-2441, incomparison with the anti-Ang2 antibodies <ANG-2>Ang2i_LC06 and<ANG-2>Ang2k_LC08

FIG. 17 Schematic representation of human Angiopoietin interaction ELISA

FIG. 18 VEGF-induced HUVEC proliferation of<VEGF-ANG-2>TvAb-2441-bevacizumab-LC06 and<VEGF-ANG-2>TvAb-2441-bevacizumab-LC08 and bevacizumab

FIG. 19 In vivo anti-angiogenic efficacy of bispecific antibody<VEGF-ANG-2>bevacizumab-LC06 antibody in comparison to <ANG-2>ANG2i-LC06, and the combination of <ANG-2> ANG2i-LC06 and bevacizumab inCalu3 xenograft model monitored via labeled anti-CD31 antibody and therelative change of CD31 signal during therapy.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to a bispecific antibody that bindsspecifically to human vascular endothelial growth factor (VEGF) andhuman angiopoietin-2 (ANG-2), said antibody comprising a firstantigen-binding site that specifically binds to human VEGF and a secondantigen-binding site that specifically binds to human ANG-2.

In an embodiment of the present invention, the bispecific antibody ischaracterized in that

-   -   i) the antibody comprises a first antigen-binding site that        specifically binds to human VEGF and a second antigen-binding        site that specifically binds to human ANG-2, and each        antigen-binding site comprises an antibody heavy chain variable        domain and an antibody light chain variable domain;    -   ii) said first antigen-binding site comprises in the heavy chain        variable domain: a CDR3 region having an amino acid sequence        selected from the group consisting of: SEQ ID NO: 1, SEQ ID NO:        9, SEQ ID NO: 17, and SEQ ID NO: 94; a CDR2 region having an        amino acid sequence selected from the group consisting of: SEQ        ID NO: 2, SEQ ID NO: 10, SEQ ID NO: 18, and SEQ ID NO: 95; and a        CDR1 region having an amino acid sequence selected from the        group consisting of: SEQ ID NO:3, SEQ ID NO: 11, SEQ ID NO: 19,        and SEQ ID NO: 96, and in the light chain variable domain:        -   a CDR3 region having an amino acid sequence selected from            the group consisting of: SEQ ID NO: 4, SEQ ID NO: 12, SEQ ID            NO: 20, and SEQ ID NO: 97,        -   a CDR2 region having an amino acid sequence selected from            the group consisting of: SEQ ID NO:5, SEQ ID NO: 13, SEQ ID            NO: 21, and SEQ ID NO: 98; and        -   a CDR1 region having an amino acid sequence selected from            the group consisting of: SEQ ID NO:6, SEQ ID NO: 14, SEQ ID            NO: 22, and SEQ ID NO: 99; and    -   iii) said second antigen-binding site comprises        -   in the heavy chain variable domain:            -   a CDR3 region having an amino acid sequence selected                from the group consisting of: SEQ ID NO: 25, SEQ ID NO:                38, SEQ ID NO: 46, SEQ ID NO: 54, SEQ ID NO: 62, SEQ ID                NO: 70, SEQ ID NO: 78, and SEQ ID NO: 86;            -   a CDR2 region having an amino acid sequence selected                from the group consisting of: SEQ ID NO: 26, SEQ ID NO:                39, SEQ ID NO: 47, SEQ ID NO: 55, SEQ ID NO: 63, SEQ ID                NO: 71, SEQ ID NO: 79, and SEQ ID NO: 87; and            -   a CDR1 region having an amino acid sequence selected                from the group consisting of: SEQ ID NO:27, SEQ ID NO:                40, SEQ ID NO: 48, SEQ ID NO: 56, SEQ ID NO: 64, SEQ ID                NO: 72, SEQ ID NO: 80, and SEQ ID NO: 88; and in the                light chain variable domain:            -   a CDR3 region having an amino acid sequence selected                from the group consisting of: SEQ ID NO: 28, SEQ ID NO:                28 with the mutations T92L, H93Q and W94T, SEQ ID NO:                41, SEQ ID NO: 49, SEQ ID NO: 57, SEQ ID NO: 65, SEQ ID                NO: 73, SEQ ID NO: 81, and SEQ ID NO: 89;            -   a CDR2 region having an amino acid sequence selected                from the group consisting of: SEQ ID NO:29, SEQ ID NO:                42, SEQ ID NO: 50, SEQ ID NO: 58, SEQ ID NO: 66, SEQ ID                NO: 74, SEQ ID NO: 82 and SEQ ID NO: 90; and            -   a CDR1 region having an amino acid sequence selected                from the group consisting of: SEQ ID NO:30, SEQ ID NO:                43, SEQ ID NO: 51, SEQ ID NO: 59, SEQ ID NO: 67, SEQ ID                NO: 75, SEQ ID NO: 83, and SEQ ID NO: 91.

In one embodiment of the invention the bispecific antibody according tothe invention is characterized in that

-   -   i) said antigen-binding sites each comprise an antibody heavy        chain variable domain and an antibody light chain variable        domain;    -   ii) said first antigen-binding site comprises, in the heavy        chain variable domain, a CDR3 region of SEQ ID NO: 1, a CDR2        region of SEQ ID NO: 2, and a CDR1 region of SEQ ID NO:3, and,        in the light chain variable domain, a CDR3 region of SEQ ID NO:        4, a CDR2 region of SEQ ID NO:5, and a CDR1 region of SEQ ID        NO:6;        -   or said first antigen-binding site comprises, in the heavy            chain variable domain, a CDR3 region of SEQ ID NO: 9, a CDR2            region of SEQ ID NO: 10, and a CDR1 region of SEQ ID NO:11,            and, in the light chain variable domain, a CDR3 region of            SEQ ID NO: 12, a CDR2 region of SEQ ID NO:13, and a CDR1            region of SEQ ID NO:14;        -   or said first antigen-binding site comprises, in the heavy            chain variable domain, a CDR3 region of SEQ ID NO: 17, a            CDR2 region of SEQ ID NO: 18, and a CDR1 region of SEQ ID            NO:19, and, in the light chain variable domain, a CDR3            region of SEQ ID NO: 20, a CDR2 region of SEQ ID NO:21, and            a CDR1 region of SEQ ID NO:22; and    -   iii) said second antigen-binding sites comprises, in the heavy        chain variable domain, a CDR3 region of SEQ ID NO: 25, a CDR2        region of SEQ ID NO: 26, and a CDR1 region of SEQ ID NO:27, and,        in the light chain variable domain, a CDR3 region of SEQ ID NO:        28 or SEQ ID NO: 28 with the mutations T92L, H93Q and W94T, a        CDR2 region of SEQ ID NO:29, and a CDR1 region of SEQ ID NO:30.

In one embodiment of the invention the bispecific antibody according tothe invention is characterized in that

-   -   i) said antigen-binding sites are each a pair of an antibody        heavy chain variable domain and an antibody light chain variable        domain;    -   ii) said first antigen-binding site comprises, as the heavy        chain variable domain, SEQ ID NO: 7, SEQ ID NO: 15, SEQ ID NO:        23, or SEQ ID NO: 100, and, as the light chain variable domain,        SEQ ID NO: 8, SEQ ID NO: 16, SEQ ID NO: 24, or SEQ ID NO: 101,        and    -   iii) said second antigen-binding sites comprises, as the heavy        chain variable domain, SEQ ID NO: 31, SEQ ID NO: 44, SEQ ID NO:        52, SEQ ID NO: 60, SEQ ID NO: 68, SEQ ID NO: 76, SEQ ID NO: 84        or SEQ ID NO: 92, and, as the light chain variable domain, SEQ        ID NO: 32, SEQ ID NO: 32 with the mutations T92L, H93Q and W94T        SEQ ID NO: 45, SEQ ID NO: 53, SEQ ID NO: 61, SEQ ID NO: 69, SEQ        ID NO: 77, SEQ ID NO: 85 or SEQ ID NO: 93.

In one embodiment of the invention the bispecific antibody according tothe invention is characterized in that said first antigen-binding sitecomprises, in the heavy chain variable domain, a CDR3 region of SEQ IDNO: 1, or SEQ ID NO: 94, a CDR2 region of SEQ ID NO: 2, or SEQ ID NO:95, and a CDR1 region of SEQ ID NO:3, or SEQ ID NO: 96, and, in thelight chain variable domain, a CDR3 region of SEQ ID NO: 4, or SEQ IDNO: 97, a CDR2 region of SEQ ID NO:5, or SEQ ID NO: 98, and a CDR1region of SEQ ID NO:6, or SEQ ID NO: 99; said second antigen-bindingsite comprises, in the heavy chain variable domain, a CDR3 region of SEQID NO: 38, SEQ ID NO: 46, SEQ ID NO: 54, SEQ ID NO: 62, SEQ ID NO: 70,SEQ ID NO: 78, or SEQ ID NO: 86, a CDR2 region of SEQ ID NO: 39, SEQ IDNO: 47, SEQ ID NO: 55, SEQ ID NO: 63, SEQ ID NO: 71, SEQ ID NO: 79, orSEQ ID NO: 87, and a CDR1 region of SEQ ID NO: 40, SEQ ID NO: 48, SEQ IDNO: 56, SEQ ID NO: 64, SEQ ID NO: 72, SEQ ID NO: 80, or SEQ ID NO: 88,and, in the light chain variable domain, a CDR3 region of SEQ ID NO: 41,SEQ ID NO: 49, SEQ ID NO: 57, SEQ ID NO: 65, SEQ ID NO: 73, SEQ ID NO:81, or SEQ ID NO: 89, a CDR2 region of SEQ ID NO: 42, SEQ ID NO: 50, SEQID NO: 58, SEQ ID NO: 66, SEQ ID NO: 74, SEQ ID NO: 82 or SEQ ID NO: 90,and a CDR1 region of SEQ ID NO: 43, SEQ ID NO: 51, SEQ ID NO: 59, SEQ IDNO: 67, SEQ ID NO: 75, SEQ ID NO: 83, or SEQ ID NO: 91.

In one embodiment of the invention the bispecific antibody according tothe invention is characterized in that said first antigen-binding sitecomprises, as the heavy chain variable domain, SEQ ID NO: 7, or SEQ IDNO: 100, and, as the light chain variable domain, SEQ ID NO: 8 or SEQ IDNO: 101, and said second antigen-binding site specifically binding toANG-2 comprises, as the heavy chain variable domain, SEQ ID NO: 44, SEQID NO: 52, SEQ ID NO: 60, SEQ ID NO: 68, SEQ ID NO: 76, SEQ ID NO: 84 orSEQ ID NO: 92, and, as the light chain variable domain, SEQ ID NO: 45,SEQ ID NO: 53, SEQ ID NO: 61, SEQ ID NO: 69, SEQ ID NO: 77, SEQ ID NO:85 or SEQ ID NO: 93.

In one embodiment of the invention the bispecific antibody according tothe invention is characterized in that said second antigen-binding sitecomprises, in the heavy chain variable domain, a CDR3 region of SEQ IDNO: 38, SEQ ID NO: 46, SEQ ID NO: 54, SEQ ID NO: 62, SEQ ID NO: 70, SEQID NO: 78, or SEQ ID NO: 86, a CDR2 region of SEQ ID NO: 39, SEQ ID NO:47, SEQ ID NO: 55, SEQ ID NO: 63, SEQ ID NO: 71, SEQ ID NO: 79, or SEQID NO: 87, and a CDR1 region of SEQ ID NO: 40, SEQ ID NO: 48, SEQ ID NO:56, SEQ ID NO: 64, SEQ ID NO: 72, SEQ ID NO: 80, or SEQ ID NO: 88, and,in the light chain variable domain, a CDR3 region of SEQ ID NO: 41, SEQID NO: 49, SEQ ID NO: 57, SEQ ID NO: 65, SEQ ID NO: 73, SEQ ID NO: 81,or SEQ ID NO: 89, a CDR2 region of SEQ ID NO: 42, SEQ ID NO: 50, SEQ IDNO: 58, SEQ ID NO: 66, SEQ ID NO: 74, SEQ ID NO: 82 or SEQ ID NO: 90,and a CDR1 region of SEQ ID NO: 43, SEQ ID NO: 51, SEQ ID NO: 59, SEQ IDNO: 67, SEQ ID NO: 75, SEQ ID NO: 83, or SEQ ID NO: 91.

In one embodiment of the invention the bispecific antibody according tothe invention is characterized in that said second antigen-binding sitecomprises, as the heavy chain variable domain, SEQ ID NO: 44, SEQ ID NO:52, SEQ ID NO: 60, SEQ ID NO: 68, SEQ ID NO: 76, SEQ ID NO: 84 or SEQ IDNO: 92, and, as the light chain variable domain, SEQ ID NO: 45, SEQ IDNO: 53, SEQ ID NO: 61, SEQ ID NO: 69, SEQ ID NO: 77, SEQ ID NO: 85 orSEQ ID NO: 93.

In one embodiment of the invention the bispecific antibody according tothe invention is characterized in that said first antigen-binding sitecomprises, in the heavy chain variable domain, a CDR3 region of SEQ IDNO: 1, a CDR2 region of SEQ ID NO: 2, and a CDR1 region of SEQ ID NO:3,and, in the light chain variable domain, a CDR3 region of SEQ ID NO: 4,a CDR2 region of SEQ ID NO:5, and a CDR1 region of SEQ ID NO:6; and saidsecond antigen-binding site comprises, in the heavy chain variabledomain, a CDR3 region of SEQ ID NO: 46, a CDR2 region of SEQ ID NO: 47,and a CDR1 region of SEQ ID NO: 48, and, in the light chain variabledomain, a CDR3 region of SEQ ID NO: 49, a CDR2 region of SEQ ID NO: 50,and a CDR1 region of SEQ ID NO: 51.

In one embodiment of the invention the bispecific antibody according tothe invention is characterized in that said first antigen-binding sitecomprises, as the heavy chain variable domain, SEQ ID NO: 7, and, as thelight chain variable domain, SEQ ID NO: 8, and said secondantigen-binding site comprises, as the heavy chain variable domain, SEQID NO: 52, and, as the light chain variable domain, SEQ ID NO: 53.

In one embodiment of the invention the bispecific antibody according tothe invention is characterized in that said first antigen-binding sitecomprises, in the heavy chain variable domain, a CDR3 region of SEQ IDNO: 1, or SEQ ID NO: 94, a CDR2 region of SEQ ID NO: 2, or SEQ ID NO:95, and a CDR1 region of SEQ ID NO:3, or SEQ ID NO: 96, and, in thelight chain variable domain, a CDR3 region of SEQ ID NO: 4, or SEQ IDNO: 97, a CDR2 region of SEQ ID NO:5, or SEQ ID NO: 98, and a CDR1region of SEQ ID NO:6, or SEQ ID NO: 99; said second antigen-bindingsites comprises, in the heavy chain variable domain, a CDR3 region ofSEQ ID NO: 62, or SEQ ID NO: 86, a CDR2 region of SEQ ID NO: 63, or SEQID NO: 87, and a CDR1 region of SEQ ID NO: 64, or SEQ ID NO: 88, and, inthe light chain variable domain, a CDR3 region of SEQ ID NO: 65, or SEQID NO: 89, a CDR2 region of SEQ ID NO: 66, or SEQ ID NO: 90, and a CDR1region of SEQ ID NO: 67, or SEQ ID NO: 91.

In one embodiment of the invention the bispecific antibody according tothe invention is characterized in that said first antigen-binding sitecomprises, as the heavy chain variable domain, SEQ ID NO: 7, or SEQ IDNO: 100, and as light chain variable domain SEQ ID NO: 8 or SEQ ID NO:101, and said second antigen-binding site comprises, as the heavy chainvariable domain, SEQ ID NO: 68 or SEQ ID NO: 92, and, as the light chainvariable domain, SEQ ID NO: 69 or SEQ ID NO: 93.

In one embodiment of the invention the bispecific antibody according tothe invention is characterized in that said first antigen-binding sitecomprises, in the heavy chain variable domain, a CDR3 region of SEQ IDNO: 1, a CDR2 region of SEQ ID NO: 2, and a CDR1 region of SEQ ID NO:3,and, in the light chain variable domain, a CDR3 region of SEQ ID NO: 4,a CDR2 region of SEQ ID NO:5, and a CDR1 region of SEQ ID NO:6; saidsecond antigen-binding site comprises, in the heavy chain variabledomain. a CDR3 region of SEQ ID NO: 62, a CDR2 region of SEQ ID NO: 63,and a CDR1 region of SEQ ID NO: 64, and, in the light chain variabledomain, a CDR3 region of SEQ ID NO: 65, a CDR2 region of SEQ ID NO: 66,and a CDR1 region of SEQ ID NO: 67.

In one embodiment of the invention the bispecific antibody according tothe invention is characterized in that said first antigen-binding sitecomprises, as the heavy chain variable domain, SEQ ID NO: 7, and, as thelight chain variable domain, SEQ ID NO: 8; and said secondantigen-binding site comprises, as the heavy chain variable domain, SEQID NO: 68, and, as the light chain variable domain, SEQ ID NO: 69.

In one embodiment of the invention the bispecific antibody according tothe invention is characterized in that said first antigen-binding site scomprises, in the heavy chain variable domain, a CDR3 region of SEQ IDNO: 1, a CDR2 region of SEQ ID NO: 2, and a CDR1 region of SEQ ID NO:3,and, in the light chain variable domain, a CDR3 region of SEQ ID NO: 4,a CDR2 region of SEQ ID NO:5, and a CDR1 region of SEQ ID NO:6; and saidsecond antigen-binding site comprises, in the heavy chain variabledomain, a CDR3 region of SEQ ID NO: 78, a CDR2 region of SEQ ID NO: 79,and a CDR1 region of SEQ ID NO: 80, and, in the light chain variabledomain a CDR3 region of SEQ ID NO: 81, a CDR2 region of SEQ ID NO: 82,and a CDR1 region of SEQ ID NO: 83.

In one embodiment of the invention the bispecific antibody according tothe invention is characterized in that said first antigen-binding sitecomprises, as the heavy chain variable domain, SEQ ID NO: 7, and, as thelight chain variable domain, SEQ ID NO: 8; and said secondantigen-binding sites comprises, as the heavy chain variable domain, SEQID NO: 84, and, as the light chain variable domain, SEQ ID NO: 85.

Another embodiment of the invention is a bispecific antibody thatspecifically binds to human vascular endothelial growth factor (VEGF)and human angiopoietin-2 (ANG-2) characterized in that the parentanti-ANG-2 antibody does not specifically bind to human Angiopoetin 1(ANG-1). Typical parent antibodies which specifically bind to humanANG-2, but not to human ANG-1 are e.g. Ang2s_R3_LC03, Ang2s_LC09,Ang2i_LC06, Ang2i_LC07, and preferably Ang2i_LC10 or antibodies bindingto the same epitope as Ang2s_R3_LC03, Ang2s_LC09, Ang2i_LC06,Ang2i_LC07, Ang2i_LC10, preferably antibodies binding to the sameepitope as Ang2i_LC06, or Ang2i_LC10. Therefore in one embodiment of theinvention the bispecific antibody that specifically binds to humanvascular endothelial growth factor (VEGF) and human angiopoietin-2(ANG-2) but not to human ANG-1 (or wherein the parent anti-ANG-2antibody does not specifically bind to human Angiopoetin 1 (ANG-1))binds to the same epitope as Ang2s_R3_LC03, Ang2s_LC09, Ang2i_LC06,Ang2i_LC07, Ang2i_LC10, preferably to the same epitope as Ang2i_LC06 orAng2i_LC10. Such bispecific antibodies that bind specifically to humanvascular endothelial growth factor (VEGF) and human angiopoietin-2(ANG-2) but not to human ANG-1 (or wherein the parent anti-ANG-2antibody does not specifically bind to human Angiopoetin 1 (ANG-1)) canhave improved properties such as e.g. biological or pharmacologicalactivity, less toxicity, or pharmacokinetic profile, compared tobispecific antibodies that specifically bind to human vascularendothelial growth factor (VEGF) and human angiopoietin-2 (ANG-2) aswell as to human ANG-1.

Thus a preferred embodiment is a bispecific antibody that specificallybinds to human VEGF and human ANG-2 which comprises a firstantigen-binding site that specifically binds to human VEGF and a secondantigen-binding site that specifically binds to human ANG-2,characterized in that the second antigen-binding site does notspecifically bind to human Angiopoetin 1 (ANG-1).

One embodiment of the invention is a bispecific antibody thatspecifically binds to human vascular endothelial growth factor (VEGF)and human angiopoietin-2 (ANG-2) which comprises a first antigen-bindingsite that specifically binds to human VEGF and a second antigen-bindingsite that specifically binds to human ANG-2, characterized in that theratio of the binding affinities K_(D) (antigen-binding site specific forVEGF)/K_(D) (antigen-binding site specific for ANG-2) is 1.0-10.0,preferably 1.5-8.0 (In one embodiment 5.0-8.0) and preferably theabsolute K_(D) value is in the range of 10⁻⁸-10⁻¹³ mol/l. K_(D) valuesare determined in a ANG-2/VEGF binding BIACORE (see Example 2, and FIG.15A). As both proteins human VEGF and human ANG-2 are present as solublereceptor ligands in human serum at approximately the sameconcentrations, blocking of both receptor ligands by a bispecificantibody characterized as described above can lead to improvedproperties with respect to the anti-angiogenic effects, tumor growthinhibition or resistance mechanism during the treatment of cancer orvascular diseases with such a bispecific antibody. Preferably saidbispecific antibody comprises a first antigen-binding site thatspecifically binds to VEGF and has a heavy chain variable domain of SEQID NO: 7 and a light chain variable domain of SEQ ID NO: 8 and a secondantigen-binding site that binds specifically to ANG-2 and has: a) eithera heavy chain variable domain of SEQ ID NO: 52 and a light chainvariable domain of SEQ ID NO: 53 or b) a heavy chain variable domain ofSEQ ID NO: 84 and a light chain variable domain of SEQ ID NO: 85.

As used herein, “antibody” refers to a binding protein that comprisesantigen-binding sites. The terms “binding site” or “antigen-bindingsite” as used herein denote the region(s) of an antibody molecule towhich a ligand actually binds. The term “antigen-binding site” includesantibody heavy chain variable domains (VH) and/or an antibody lightchain variable domains (VL), or pairs of VH/VL, and can be derived fromwhole antibodies or antibody fragments such as single chain Fv, a VHdomain and/or a VL domain, Fab, or (Fab)2. In one embodiment of thecurrent invention, each of the antigen-binding sites comprises anantibody heavy chain variable domain (VH) and/or an antibody light chainvariable domain (VL), and preferably is formed by a pair consisting ofan antibody light chain variable domain (VL) and an antibody heavy chainvariable domain (VH).

The antigen-binding site, and especially heavy chain variable domains(VH) and/or antibody light chain variable domains (VL), thatspecifically bind to human vascular endothelial growth factor (VEGF) canbe derived a) from known anti-VEGF antibodies such as Kim et al., Nature362 (1993) 841-844; Warren, R. S., et al., J. Clin. Invest. 95 (1995)1789-1797; Borgstrom, P., et al., Cancer Res. 56 (1996) 4032-4039;Melnyk, O., et al., Cancer Res. 56 (1996) 921-924). WO 94/10202, WO98/45332, WO 2005/00900, WO 00/35956 and US 2007/0141065 orb) from newanti-VEGF antibodies obtained by de novo immunization methods usinginter alia either the human VEGF protein or nucleic acid or fragmentsthereof or by phage display.

The antigen-binding site, and especially heavy chain variable domains(VH) and/or antibody light chain variable domains (VL), thatspecifically bind to human angiopoietin-2 (ANG-2) can be derived a) fromknown anti-ANG-2 antibodies such as WO 03/030833, WO 2006/068953, WO2006/045049 or U.S. Pat. No. 6,166,185; orb) from new anti-ANG-2antibodies obtained e.g. by de novo immunization methods using interalia either the human ANG-2 protein or nucleic acid or fragments thereofor by phage display.

Antibody specificity refers to selective recognition of the antibody fora particular epitope of an antigen. Natural antibodies, for example, aremonospecific.

“Bispecific antibodies” according to the invention are antibodies whichhave two different antigen-binding specificities. Where an antibody hasmore than one specificity, the recognized epitopes may be associatedwith a single antigen or with more than one antigen. Antibodies of thepresent invention are specific for two different antigens, i.e. VEGF asfirst antigen and ANG-2 as second antigen.

The term “monospecific” antibody as used herein denotes an antibody thathas one or more binding sites, each of which bind to the same epitope ofthe same antigen.

The term “valent” as used within the current application denotes thepresence of a specified number of binding sites in an antibody molecule.As such, the terms “bivalent”, “tetravalent”, and “hexavalent” denotethe presence of two binding sites, four binding sites, and six bindingsites, respectively, in an antibody molecule. The bispecific antibodiesaccording to the invention are at least “bivalent” and may be“trivalent” or “multivalent” (e.g. (“tetravalent” or “hexavalent”).Preferably the bispecific antibody according to the invention isbivalent, trivalent or tetravalent. In one embodiment said bispecificantibody is bivalent. In one embodiment said bispecific antibody istrivalent. In one embodiment said bispecific antibody is tetravalent.

Antibodies of the present invention have two or more binding sites andare bispecific. That is, the antibodies may be bispecific even in caseswhere there are more than two binding sites (i.e. that the antibody istrivalent or multivalent). Bispecific antibodies of the inventioninclude, for example, multivalent single chain antibodies, diabodies andtriabodies, as well as antibodies having the constant domain structureof full length antibodies to which further antigen-binding sites (e.g.,single chain Fv, a VH domain and/or a VL domain, Fab, or (Fab)2,) arelinked via one or more peptide-linkers. The antibodies can be fulllength from a single species, or be chimerized or humanized. For anantibody with more than two antigen binding sites, some binding sitesmay be identical, so long as the protein has binding sites for twodifferent antigens. That is, whereas a first binding site is specificfor a VEGF, a second binding site is specific for ANG-2, and vice versa.

Human vascular endothelial growth factor (VEGF/VEGF-A) (SEQ ID No: 105)is described in e.g. Leung, D. W., et al., Science 246 (1989) 1306-9;Keck, P. J., et al., Science 246 (1989) 1309-12 and Connolly, D. T., etal., J. Biol. Chem. 264 (1989) 20017-24. VEGF is involved in theregulation of normal and abnormal angiogenesis and neovascularizationassociated with tumors and intraocular disorders (Ferrara, N., et al.,Endocr. Rev. 18 (1997) 4-25; Berkman, R. A., et al., J. Clin. Invest. 91(1993) 153-159; Brown, L. F., et al., Human Pathol. 26 (1995) 86-91;Brown, L. F., et al., Cancer Res. 53 (1993) 4727-4735; Mattern, J., etal., Brit. J. Cancer. 73 (1996) 931-934; and Dvorak, H., et al., Am. J.Pathol. 146 (1995) 1029-1039). VEGF is a homodimeric glycoprotein thathas been isolated from several sources. VEGF shows highly specificmitogenic activity for endothelial cells.

Human angiopoietin-2 (ANG-2) (alternatively abbreviated with ANGPT2 orANG2) (SEQ ID No: 106) is described in Maisonpierre, P. C., et al,Science 277 (1997) 55-60 and Cheung, A. H., et al., Genomics 48 (1998)389-91. The angiopoietins-1 and -2 (ANG-1 (SEQ ID No: 107) and ANG-2(SEQ ID No: 106)) were discovered as ligands for the Ties, a family oftyrosine kinases that is selectively expressed within the vascularendothelium. Yancopoulos, G. D., et al., Nature 407 (2000) 242-48. Thereare now four definitive members of the angiopoietin family.Angiopoietin-3 and -4 (Ang-3 and Ang-4) may represent widely divergedcounterparts of the same gene locus in mouse and man. Kim, I., et al.,FEBS Let, 443 (1999) 353-56; Kim, I., et al., J Biol Chem 274 (1999)26523-28. ANG-1 and ANG-2 were originally identified in tissue cultureexperiments as agonist and antagonist, respectively (see for ANG-1:Davis, S., et al., Cell 87 (1996) 1161-69; and for ANG-2: Maisonpierre,P. C., et al., Science 277 (1997) 55-60) All of the known angiopoietinsbind primarily to Tie2, and both Ang-1 and -2 bind to Tie2 with anaffinity of 3 nM (K_(D)). Maisonpierre, P. C., et al., Science 277(1997) 55-60.

An antigen-binding site of an antibody of the invention can contain sixcomplementarity determining regions (CDRs) which contribute in varyingdegrees to the affinity of the binding site for antigen. There are threeheavy chain variable domain CDRs (CDRH1, CDRH2 and CDRH3) and threelight chain variable domain CDRs (CDRL1, CDRL2 and CDRL3). The extent ofCDR and framework regions (FRs) is determined by comparison to acompiled database of amino acid sequences in which those regions havebeen defined according to variability among the sequences. Also includedwithin the scope of the invention are functional antigen binding sitescomprised of fewer CDRs (i.e., where binding specificity is determinedby three, four or five CDRs). For example, less than a complete set of 6CDRs may be sufficient for binding. In some cases, a VH or a VL domainwill be sufficient.

In certain embodiments, antibodies of the invention further compriseimmunoglobulin constant regions of one or more immunoglobulin classes.Immunoglobulin classes include IgG, IgM, IgA, IgD, and IgE isotypes and,in the case of IgG and IgA, their subtypes. In a preferred embodiment,an antibody of the invention has a constant domain structure of an IgGtype antibody, but has four antigen binding sites. This is accomplishede.g. by linking two complete antigen binding sites (e.g., a single chainFv) specifically binding to VEGF to either to N- or C-terminus heavy orlight chain of a full antibody specifically binding to ANG-2.Alternatively this is accomplished by fusing two complete bindingpeptides specifically binding to ANG-2 to either to C-terminus heavychain of a full antibody specifically binding to VEGF. The fourantigen-binding sites preferably comprise two antigen-binding sites foreach of two different binding specificities.

The terms “monoclonal antibody” or “monoclonal antibody composition” asused herein refer to a preparation of antibody molecules of a singleamino acid composition.

The term “chimeric antibody” refers to an antibody comprising a variableregion, i.e., binding region, from one source or species and at least aportion of a constant region derived from a different source or species,usually prepared by recombinant DNA techniques. Chimeric antibodiescomprising a murine variable region and a human constant region arepreferred. Other preferred forms of “chimeric antibodies” encompassed bythe present invention are those in which the constant region has beenmodified or changed from that of the original antibody to generate theproperties according to the invention, especially in regard to C1qbinding and/or Fc receptor (FcR) binding. Such chimeric antibodies arealso referred to as “class-switched antibodies.”. Chimeric antibodiesare the product of expressed immunoglobulin genes comprising DNAsegments encoding immunoglobulin variable regions and DNA segmentsencoding immunoglobulin constant regions. Methods for producing chimericantibodies involve conventional recombinant DNA and gene transfectiontechniques are well known in the art. See, e.g., Morrison, S. L., etal., Proc. Natl. Acad. Sci. USA 81 (1984) 6851-6855; U.S. Pat. Nos.5,202,238 and 5,204,244.

The term “humanized antibody” refers to antibodies in which theframework or “complementarity determining regions” (CDR) have beenmodified to comprise the CDR of an immunoglobulin of differentspecificity as compared to that of the parent immunoglobulin. In apreferred embodiment, a murine CDR is grafted into the framework regionof a human antibody to prepare the “humanized antibody.” See, e.g.,Riechmann, L., et al., Nature 332 (1988) 323-327; and Neuberger, M. S.,et al., Nature 314 (1985) 268-270. Particularly preferred CDRscorrespond to those representing sequences recognizing the antigensnoted above for chimeric antibodies. Other forms of “humanizedantibodies” encompassed by the present invention are those in which theconstant region has been additionally modified or changed from that ofthe original antibody to generate the properties according to theinvention, especially in regard to C1q binding and/or Fc receptor (FcR)binding.

The term “human antibody”, as used herein, is intended to includeantibodies having variable and constant regions derived from human germline immunoglobulin sequences. Human antibodies are well-known in thestate of the art (van Dijk, M. A., and van de Winkel, J. G., Curr. Opin.Chem. Biol. 5 (2001) 368-374). Human antibodies can also be produced intransgenic animals (e.g., mice) that are capable, upon immunization, ofproducing a full repertoire or a selection of human antibodies in theabsence of endogenous immunoglobulin production. Transfer of the humangerm-line immunoglobulin gene array in such germ-line mutant mice willresult in the production of human antibodies upon antigen challenge(see, e.g., Jakobovits, A., et al., Proc. Natl. Acad. Sci. USA 90 (1993)2551-2555; Jakobovits, A., et al., Nature 362 (1993) 255-258;Bruggemann, M., et al., Year Immunol. 7 (1993) 33-40). Human antibodiescan also be produced in phage display libraries (Hoogenboom, H. R., andWinter, G., J. Mol. Biol. 227 (1992) 381-388; Marks, J. D., et al., J.Mol. Biol. 222 (1991) 581-597). The techniques of Cole, A., et al. andBoerner, P., et al. are also available for the preparation of humanmonoclonal antibodies (Cole, A., et al., Monoclonal Antibodies andCancer Therapy, Liss, A. L., p. 77 (1985); and Boerner, P., et al., J.Immunol. 147 (1991) 86-95). As already mentioned for chimeric andhumanized antibodies according to the invention the term “humanantibody” as used herein also comprises such antibodies which aremodified in the constant region to generate the properties according tothe invention, especially in regard to C1q binding and/or FcR binding,e.g. by “class switching” i.e. change or mutation of Fc parts (e.g. fromIgG1 to IgG4 and/or IgG1/IgG4 mutation).

The term “recombinant human antibody”, as used herein, is intended toinclude all human antibodies that are prepared, expressed, created orisolated by recombinant means, such as antibodies isolated from a hostcell such as a NS0 or CHO cell or from an animal (e.g. a mouse) that istransgenic for human immunoglobulin genes or antibodies expressed usinga recombinant expression vector transfected into a host cell. Suchrecombinant human antibodies have variable and constant regions in arearranged form. The recombinant human antibodies according to theinvention have been subjected to in vivo somatic hypermutation. Thus,the amino acid sequences of the VH and VL regions of the recombinantantibodies are sequences that, while derived from and related to humangerm line VH and VL sequences, may not naturally exist within the humanantibody germ line repertoire in vivo.

The term “variable domain”, when used in reference to a domain of aheavy chain or a light chain, refer respectively to the portion of aheavy chain or a light chain which is involved directly in binding theantibody to the antigen. The variable domains of human light and heavychains have the same general structure and each domain comprises fourframework (FR) regions whose sequences are widely conserved, connectedby three “hypervariable regions” (or complementarity determiningregions, CDRs). The framework regions adopt a β-sheet conformation andthe CDRs may form loops connecting the β-sheet structure. The CDRs ineach chain are held in their three-dimensional structure by theframework regions and form together with the CDRs from the other chainthe antigen binding site. The antibody heavy and light chain CDR3regions play a particularly important role in the bindingspecificity/affinity of the antibodies according to the invention andtherefore provide a further object of the invention.

The terms “hypervariable region” or “antigen-binding portion of anantibody”, when used herein, refer to the amino acid residues of anantibody which are responsible for antigen-binding. The hypervariableregion comprises amino acid residues from the “complementaritydetermining regions” or “CDRs”. “Framework” or “FR” regions are thosevariable domain regions other than the hypervariable region residues asherein defined. Therefore, the light and heavy chains of an antibodycomprise from N- to C-terminus the domains FR1, CDR1, FR2, CDR2, FR3,CDR3, and FR4. CDRs on each chain are separated by such framework aminoacids. Especially, CDR3 of the heavy chain is the region whichcontributes most to antigen binding. CDR and FR regions are determinedaccording to the standard definition of Kabat et al., Sequences ofProteins of Immunological Interest, 5th ed., Public Health Service,National Institutes of Health, Bethesda, Md. (1991).

The bispecific antibodies according to the invention include, inaddition, such antibodies having “conservative sequence modifications”(which is meant by “variants” of the bispecific antibodies). This meansnucleotide and amino acid sequence modifications which do not affect oralter the above-mentioned characteristics of the antibody according tothe invention. Modifications can be introduced by standard techniquesknown in the art, such as site-directed mutagenesis and PCR-mediatedmutagenesis. Conservative amino acid substitutions include ones in whichthe amino acid residue is replaced with an amino acid residue having asimilar side chain. Families of amino acid residues having similar sidechains have been defined in the art. These families include amino acidswith basic side chains (e.g. lysine, arginine, histidine), acidic sidechains (e.g. aspartic acid, glutamic acid), uncharged polar side chains(e.g. glycine, asparagine, glutamine, serine, threonine, tyrosine,cysteine, tryptophan), nonpolar side chains (e.g. alanine, valine,leucine, isoleucine, proline, phenylalanine, methionine), beta-branchedside chains (e.g. threonine, valine, isoleucine) and aromatic sidechains (e.g. tyrosine, phenylalanine, tryptophan, histidine). Thus, apredicted nonessential amino acid residue in a bispecific <VEGF-ANG-2>antibody can be preferably replaced with another amino acid residue fromthe same side chain family. A “variant” bispecific <VEGF-ANG-2>antibody, refers therefore herein to a molecule which differs in aminoacid sequence from a “parent” bispecific <VEGF-ANG-2> antibody aminoacid sequence by up to ten, preferably from about two to about five,additions, deletions and/or substitutions in one or more variable regionor constant region of the parent antibody. Amino acid substitutions canbe performed by mutagenesis based upon molecular modeling as describedby Riechmann, L., et al., Nature 332 (1988) 323-327 and Queen, C., etal., Proc. Natl. Acad. Sci. USA 86 (1989) 10029-10033. A “variant”bispecific <VEGF-ANG-2> antibody according to the invention includesalso bispecific antibodies formats in which the linker (if existing) wasmodified, or replaced by another linker.

As used herein, the term “binding” or “specifically binding” refers tothe binding of the antibody to an epitope of the antigen (either humanVEGF or human ANG-2) in an in vitro assay, preferably in an plasmonresonance assay (BIAcore, GE-Healthcare Uppsala, Sweden) (Example 2)with purified wild-type antigen. The affinity of the binding is definedby the terms K_(A) (rate constant for the association of the antibodyfrom the antibody/antigen complex), K_(D) (dissociation constant), andK_(D) (K_(D)/K_(A)). Binding or specifically binding means a bindingaffinity (K_(D)) of 10⁻⁸ mol/l or less, preferably 10⁻⁹ M to 10⁻¹³mol/l.

Binding of the antibody to the FcγRIII can be investigated by a BIAcoreassay (GE-Healthcare Uppsala, Sweden). The affinity of the binding isdefined by the terms K_(A) (rate constant for the association of theantibody from the antibody/antigen complex), K_(D) (dissociationconstant), and K_(D) (K_(D)/K_(A)).

As used herein, the term “not binding to ANG-1” or “not specificallybinding to ANG-1” denotes that the antibody has an EC50-value above 8000ng/ml in an in-vitro ANG-1 binding ELISA assay (according to Example 9).

The term “epitope” includes any polypeptide determinant capable ofspecific binding to an antibody. In certain embodiments, epitopedeterminants include chemically active surface groupings of moleculessuch as amino acids, sugar side chains, phosphoryl, or sulfonyl, and, incertain embodiments, may have specific three dimensional structuralcharacteristics, and or specific charge characteristics. An epitope is aregion of an antigen that is bound by an antibody.

In certain embodiments, an antibody is said to specifically bind anantigen when it preferentially recognizes its target antigen in acomplex mixture of proteins and/or macromolecules.

In one embodiment of the invention the bispecific antibody comprises afull length parent antibody as scaffold.

The term “full length antibody” denotes an antibody consisting of two“full length antibody heavy chains” and two “full length antibody lightchains” A “full length antibody heavy chain” is a polypeptide consistingin N-terminal to C-terminal direction of an antibody heavy chainvariable domain (VH), an antibody constant heavy chain domain 1 (CH1),an antibody hinge region (HR), an antibody heavy chain constant domain 2(CH2), and an antibody heavy chain constant domain 3 (CH3), abbreviatedas VH-CH1-HR-CH2-CH3; and optionally an antibody heavy chain constantdomain 4 (CH4) in the case of an antibody of the subclass IgE.Preferably the “full length antibody heavy chain” is a polypeptideconsisting in N-terminal to C-terminal direction of VH, CH1, HR, CH2 andCH3. A “full length antibody light chain” is a polypeptide consisting inN-terminal to C-terminal direction of an antibody light chain variabledomain (VL), and an antibody light chain constant domain (CL),abbreviated as VL-CL. The antibody light chain constant domain (CL) canbe κ (kappa) or λ (lambda). The two full length antibody chains arelinked together via inter-polypeptide disulfide bonds between the CLdomain and the CH1 domain and between the hinge regions of the fulllength antibody heavy chains. Examples of typical full length antibodiesare natural antibodies like IgG (e.g. IgG 1 and IgG2), IgM, IgA, IgD,and IgE. The full length antibodies according to the invention can befrom a single species e.g. human, or they can be chimerized or humanizedantibodies. The full length antibodies according to the inventioncomprise two antigen binding sites each formed by a pair of VH and VL,which both specifically bind to the same antigen. Thus a monospecificbivalent (=full length) antibody comprising a first antigen-binding siteand consisting of two antibody light chains and two antibody heavychains is a full length antibody. The C-terminus of the heavy or lightchain of said full length antibody denotes the last amino acid at theC-terminus of said heavy or light chain. The N-terminus of the heavy orlight chain of said full length antibody denotes the last amino acid atthe N-terminus of said heavy or light chain.

A preferred embodiment for bispecific antibody formats for thebispecific antibody that specifically binds to human vascularendothelial growth factor (VEGF) and human angiopoietin-2 (ANG-2)according to the invention are bivalent antibodies with two differentspecifities as e.g. a) described in WO 2009/080251, WO 2009/080252 or WO2009/080253 (domain exchanged antibodies—see Example 13) orb) based on ascFab-Fc fusion antibody wherein one single chain Fab fragment(eventually disulfide stabilized) is specific for VEGF and the othersingle chain Fab fragment (eventually disulfide stabilized) for ANG-2(see Example 14) or c) described in Ridgway, J. B., Protein Eng. 9(1996) 617-621; WO 96/027011; Merchant, A. M., et al., Nature Biotech 16(1998) 677-681; Atwell, S., et al., J. Mol. Biol. 270 (1997) 26-35 andEP 1 870 459A1.

In one embodiment the bispecific antibody according to the inventioncomprises the amino acid sequences of SEQ ID NO: 121, SEQ ID NO: 122,SEQ ID NO: 123 and SEQ ID NO: 124 or variants thereof.

In one embodiment the bispecific antibody according to the inventioncomprises the amino acid sequences of SEQ ID NO: 125, SEQ ID NO: 126,SEQ ID NO: 127 and SEQ ID NO: 128 or variants thereof.

In one embodiment the bispecific antibody according to the inventioncomprises the amino acid sequences of SEQ ID NO: 129, SEQ ID NO: 130,SEQ ID NO: 131 and SEQ ID NO: 132 or variants thereof.

In one embodiment the bispecific antibody according to the inventioncomprises the amino acid sequences of SEQ ID NO: 133, and SEQ ID NO: 134or variants thereof.

In one embodiment the bispecific antibody according to the inventioncomprises the amino acid sequences of SEQ ID NO: 135 and SEQ ID NO: 136or variants thereof.

These amino acid sequences are based on the heavy chain variable domainsof SEQ ID NO: 7, and the light chain variable domains of SEQ ID NO: 8(derived from bevacizumab) as the binding site for VEGF, and on theheavy chain variable domains of SEQ ID NO: 52, and the light chainvariable domains of SEQ ID NO: 53 (derived from Ang2i_LC06)) as thebinding site for ANG-2.

In one embodiment said bispecific antibody is trivalent using, forexample, formats based on a full length antibody specifically binding toone of the two antigens VEGF or ANG-2, to which only at one C-terminusof one heavy chain a scFab fragment is fused which specifically binds tothe other of the two antigens VEGF or ANG-2, including knobs—into holestechnology, as described e.g. in EP Appl. No 09004909.9 (see Example 11)or, for example, formats based on a full length antibody specificallybinding to one of the two antigens VEGF or ANG-2, to which at oneC-terminus of one heavy chain a VH or VH-CH1 fragment and at the otherC-terminus of the second heavy chain a VL or VL-CL fragment is fusedwhich specifically binds to the other of the two antigens VEGF or ANG-2,including knobs—into holes technology, as described e.g. in EP Appl. No09005108.7 (see Example 12).

In one embodiment the bispecific antibody according to the invention iscomprises the amino acid sequences of SEQ ID NO: 115, SEQ ID NO: 116,and SEQ ID NO: 117 or variants thereof

In one embodiment the bispecific antibody according to the inventioncomprises the amino acid sequences of SEQ ID NO: 118, SEQ ID NO: 119,and SEQ ID NO: 120 or variants thereof.

These amino acid sequences are based on the heavy chain variable domainsof SEQ ID NO: 7, and the light chain variable domains of SEQ ID NO: 8(derived from bevacizumab) as the binding site for VEGF, and on theheavy chain variable domains of SEQ ID NO: 52, and the light chainvariable domains of SEQ ID NO: 53 (derived from Ang2i_LC06)) as thebinding site for ANG-2.

Preferred bispecific antibody formats for the bispecific antibody thatspecifically binds to human vascular endothelial growth factor (VEGF)and human angiopoietin-2 (ANG-2) according to the invention aretetravalent antibodies (TvAb) with two different specifities asdescribed e.g. in WO 2007/024715, or WO 2007/109254 or EP Appl. No09004909.9. Thus in one embodiment said bispecific antibody istetravalent using formats as described e.g. in WO 2007/024715, or WO2007/109254 or EP Appl. No 09004909.9 (see Examples 1 or 10).

In one embodiment of the invention the bispecific tetravalent antibodyTvAb-2441-bevacizumab-LC06 comprises a peptide of SEQ ID No: 102 and thelight chain of SEQ ID No: 62 or variants thereof.

In one embodiment the bispecific antibody according to the inventioncomprises the amino acid sequences of SEQ ID NO: 109 and SEQ ID NO: 110or variants thereof.

In one embodiment the bispecific antibody according to the inventioncomprises the amino acid sequences of SEQ ID NO: 111 and SEQ ID NO: 112or variants thereof.

In one embodiment the bispecific antibody according to the inventioncomprises the amino acid sequences of SEQ ID NO: 113 and SEQ ID NO: 114or variants thereof

These amino acid sequences are based on the heavy chain variable domainsof SEQ ID NO: 7, and the light chain variable domains of SEQ ID NO: 8(derived from bevacizumab) as to the binding site for VEGF, and on theheavy chain variable domains of SEQ ID NO: 52, and the light chainvariable domains of SEQ ID NO: 53 (derived from Ang2i_LC06)) as to thebinding site for ANG-2.

In one embodiment of the invention the bispecific tetravalent antibodyTvAb-2441-bevacizumab-LC08 comprises a peptide of SEQ ID No: 103 and thelight chain of SEQ ID No: 62 or variants thereof.

The binding sites in an antibody according to the invention may be eachformed by a pair of two variable domains, i.e. of one heavy chainvariable domain and one light chain variable domain. The minimal bindingsite determinant in an antibody is the heavy chain CDR3 region.

In one embodiment the bispecific antibody according to the invention istetravalent. In a further embodiment said tetravalent bispecificantibody consists of:

-   -   a) a monospecific bivalent parent antibody consisting of two        full length antibody heavy chains and two full length antibody        light chains whereby each chain comprises only one variable        domain,    -   b) two peptide-linkers, and    -   c) two monospecific monovalent single chain antibodies each        consisting of an antibody heavy chain variable domain, an        antibody light chain variable domain, and a single-chain-linker        between said antibody heavy chain variable domain and said        antibody light chain variable domain.

Preferably said single chain antibodies are linked to the same terminus(C- and N-terminus) of the monospecific bivalent antibody heavy chainsor, alternatively to the same terminus (preferably the C-terminus) ofthe monospecific bivalent antibody light chains, and more preferably tothe same terminus (C- and N-terminus) of the monospecific bivalentantibody heavy chains.

In another embodiment said bispecific antibody is tetravalent andconsists of:

-   -   a) a full length antibody comprising said antigen-binding site        and consisting of two antibody heavy chains and two antibody        light chains; and    -   b) two identical single chain Fab fragments comprising said        second antigen-binding site, wherein said single chain Fab        fragments are fused to said full length antibody via a peptide        connector at the C- or N-terminus of the heavy or light chain of        said full length antibody.

In another embodiment said bispecific antibody is tetravalent, andconsists of

-   -   a) a full length antibody comprising said second antigen-binding        site and consisting of two antibody heavy chains and two        antibody light chains; and    -   b) two identical single chain Fab fragments comprising said        first antigen-binding site, wherein said single chain Fab        fragments are fused to said full length antibody via a peptide        connector at the C- or N-terminus of the heavy or light chain of        said full length antibody.

Preferably said single chain Fab fragments under b) are fused to saidfull length antibody under a) via a peptide connector at the C-terminusof the heavy or light chain of said full length antibody.

In one embodiment the two identical single chain Fab fragments whichbind to a second antigen are fused to the full length antibody via apeptide connector at the C-terminus of each heavy or light chain of saidfull length antibody.

In one embodiment the two identical single chain Fab fragments whichbind to a second antigen are fused to the full length antibody via apeptide connector at the C-terminus of each heavy chain of said fulllength antibody.

In one embodiment the two identical single chain Fab fragments whichbind to a second antigen are fused to the full length antibody via apeptide connector at the C-terminus of each light chain of said fulllength antibody.

Such embodiments which include single chain Fab fragments are describedin more detail in e.g. EP Appl. No 09004909.9 which is incorporated byreference.

The term “peptide-linker” as used within the invention denotes a peptidewith amino acid sequences, which is preferably of synthetic origin.These peptide-linkers according to invention are used to link thedifferent antigen-binding sites and/or antibody fragments eventuallycomprising the different antigen-binding sites (e.g. single chain Fv,full length antibodies, a VH domain and/or a VL domain, Fab, (Fab)2, Fcpart) together to form a bispecific antibody according to the invention.The peptide-linkers can comprise one or more of the following amino acidsequences listed in Table 1 as well as further arbitrarily selectedamino acids. Said peptide-linkers are peptides with an amino acidsequence with a length of at least 5 amino acids, preferably of at least10 amino acids, more preferably with a length between 10 and 50 aminoacids. Preferably said peptide-linkers under b) are peptides with anamino acid sequence with a length of at least 10 amino acids. In oneembodiment said peptide-linker is (G×S)n with G=glycine, S=serine, (x=3and n=3, 4, 5 or 6) or (x=4 and n=2, 3, 4 or 5), preferably x=4 and n=2or 3, more preferably with x=4, n=2 ((G45)2). To said (G×S)npeptide-linker also additional G=glycines can be added, e.g. GG, or GGG.

The term “single-chain-linker” as used within the invention denotes apeptide with amino acid sequences, which is preferably of syntheticorigin. These single-chain-linkers according to invention are used tolink a VH and a VL domain to form a single chain Fv. Preferably thesingle-chain-linker is a peptide with an amino acid sequence with alength of at least 15 amino acids, more preferably with a length of atleast 20 amino acids. In one embodiment said single-chain-linker is(G×S)n with G=glycine, S=serine, (x=3 and n=4, 5 or 6) or (x=4 and n=3,4 or 5), preferably with x=4, n=4 or 5, more preferably with x=4, n=4.

Furthermore said single chain (single chain Fv) antibodies arepreferably disulfide stabilized. Such further disulfide stabilization ofsingle chain antibodies is achieved by the introduction of a disulfidebond between the variable domains of the single chain antibodies and isdescribed e.g in WO 94/029350, Rajagopal, V., et al., Prot. Engin. 10(12) (1997) 1453-59; Kobayashi, H., et al., Nuclear Medicine & Biology25 (1998) 387-393; or Schmidt, M., et al., Oncogene 18 (1999) 1711-1721.

In one embodiment of the disulfide stabilized single chain antibodies,the disulfide bond is independently for each single chain antibodyselected from:

-   -   i) a bond between position 44 of the heavy chain variable domain        to position 100 of the light chain variable domain,    -   ii) a bond between position 105 of the heavy chain variable        domain to position 43 of the light chain variable domain, and    -   iii) a bond between position 101 of the heavy chain variable        domain to position 100 of the light chain variable domain.

In one embodiment the disulfide bond is a bond between position 44 ofthe heavy chain variable domain to position 100 of the light chainvariable domain.

In one embodiment the disulfide bond a bond between position 105 of theheavy chain variable domain to position 43 of the light chain variabledomain.

In one embodiment of the present invention, the antibody is tetravalentand has a structure based on a full length antibody that specificallybinds to Antigen A to which two (optionally disulfide-stabilized) singlechain Fvs specifically binding to Antigen B are linked via apeptide-linker. One of Antigens A or B is VEGF and the other is ANG-2.The single chain Fvs are linked via a peptide linker to the full-lengthantibody. This embodiment is exemplified in the schemes of FIGS. 1 and2.

In one embodiment, single chain (single chain Fv) antibodies withoutsaid optional disulfide stabilization between the variable domains VHand VL of the single chain antibody (single chain Fv) are preferred.

In a further embodiment said tetravalent bispecific antibody ischaracterized in that the full-length antibody specifically binds toVEGF and the two monovalent monospecific single chain antibodies bind toANG-2.

In a further embodiment said tetravalent bispecific antibody ischaracterized in that the full-length antibody specifically binds toANG-2 and the two monovalent monospecific single chain antibodies bindto VEGF

A “single chain Fab fragment” (see FIG. 11) is a polypeptide consistingof an antibody heavy chain variable domain (VH), an antibody constantdomain 1 (CH1), an antibody light chain variable domain (VL), anantibody light chain constant domain (CL) and a linker, wherein saidantibody domains and said linker have one of the following orders inN-terminal to C-terminal direction:

-   -   a) VH-CH1-linker-VL-CL, b) VL-CL-linker-VH-CH1, c)        VH-CL-linker-VL-CH1 or d) VL-CH1-linker-VH-CL; and wherein said        linker is a polypeptide of at least 30 amino acids, preferably        between 32 and 50 amino acids. Said single chain Fab        fragments a) VH-CH1-linker-VL-CL, b) VL-CL-linker-VH-CH1, c)        VH-CL-linker-VL-CH1 and d) VL-CH1-linker-VH-CL, are stabilized        via the natural disulfide bond between the CL domain and the CH1        domain. The term “N-terminus” denotes the last amino acid of the        N-terminus. The term “C-terminus” denotes the last amino acid of        the C-terminus.

In a preferred embodiment said antibody domains and said linker in saidsingle chain Fab fragment have one of the following orders in N-terminalto C-terminal direction:

-   -   a) VH-CH1-linker-VL-CL, orb) VL-CL-linker-VH-CH1, more        preferably VL-CL-linker-VH-CH1.

In another preferred embodiment said antibody domains and said linker insaid single chain Fab fragment have one of the following orders inN-terminal to C-terminal direction:

-   -   a) VH-CL-linker-VL-CH1 or b) VL-CH1-linker-VH-CL.

The term “peptide connector” as used within the invention denotes apeptide with amino acid sequences, which is preferably of syntheticorigin. These peptide connectors according to invention are used to fusethe single chain Fab fragments to the C- or N-terminus of the fulllength antibody to form a multispecific antibody according to theinvention. Preferably said peptide connectors are peptides with an aminoacid sequence with a length of at least 5 amino acids, preferably with alength of 5 to 100, more preferably of 10 to 50 amino acids. In oneembodiment said peptide connector is (G×S)n or (G×S)nGm with G=glycine,S=serine, and (x=3, n=3, 4, 5 or 6, and m=0, 1, 2 or 3) or (x=4, n=2, 3,4 or 5 and m=0, 1, 2 or 3), preferably x=4 and n=2 or 3, more preferablywith x=4, n=2. In one embodiment said peptide connector is (G4S)2.

The term “linker” as used within the invention denotes a peptide withamino acid sequences which is preferably of synthetic origin. Thesepeptides according to invention are used to link a) VH-CH1 to VL-CL, b)VL-CL to VH-CH1, c) VH-CL to VL-CH1 or d) VL-CH1 to VH-CL to form thefollowing single chain Fab fragments according to the invention a)VH-CH1-linker-VL-CL, b) VL-CL-linker-VH-CH1, c) VH-CL-linker-VL-CH1 ord) VL-CH1-linker-VH-CL. Said linker within the single chain Fabfragments is a peptide with an amino acid sequence with a length of atleast 30 amino acids, preferably with a length of 32 to 50 amino acids.In one embodiment said linker is (G×S)n with G=glycine, S=serine, (x=3,n=8, 9 or 10 and m=0, 1, 2 or 3) or (x=4 and n=6, 7 or 8 and m=0, 1, 2or 3), preferably with x=4, n=6 or 7 and m=0, 1, 2 or 3, more preferablywith x=4, n=7 and m=2. In one embodiment said linker is (G₄S)₆G₂.

Optionally in said single chain Fab fragment, in addition to the naturaldisulfide bond between the CL-domain and the CH1 domain, the antibodyheavy chain variable domain (VH) and the antibody light chain variabledomain (VL) are disulfide stabilized by the introduction of a disulfidebond between the following positions:

-   -   i) heavy chain variable domain position 44 and light chain        variable domain position 100,    -   ii) heavy chain variable domain position 105 and light chain        variable domain position 43, or    -   iii) heavy chain variable domain position 101 and light chain        variable domain position 100 (numbering always according to EU        index of Kabat).

Such further disulfide stabilization of single chain Fab fragments isachieved by the introduction of a disulfide bond between the variabledomains VH and VL of the single chain Fab fragments. Techniques tointroduce unnatural disulfide bridges for stabilization for a singlechain Fv are described e.g. in WO 94/029350, Rajagopal, V., et al, Prot.Engin. (1997) 1453-59; Kobayashi, H., et al; Nuclear Medicine & Biology,Vol. 25, (1998) 387-393; or Schmidt, M., et al, Oncogene (1999) 18,1711-1721. In one embodiment the optional disulfide bond between thevariable domains of the single chain Fab fragments comprised in theantibody according to the invention is between heavy chain variabledomain position 44 and light chain variable domain position 100. In oneembodiment the optional disulfide bond between the variable domains ofthe single chain Fab fragments comprised in the antibody according tothe invention is between heavy chain variable domain position 105 andlight chain variable domain position 43 (numbering always according toEU index of Kabat).

In an embodiment of the present invention, single chain Fab fragmentswithout said optional disulfide stabilization between the variabledomains VH and VL of the single chain Fab fragments are preferred.

In a preferred embodiment of an tetravalent bispecific antibodyaccording to the invention, the antibody comprises two identical singlechain Fab fragments (preferably VL-CL-linker-VH-CH1) which are bothfused to the two C-termini of the two heavy chains or to the twoC-termini of the two light chains of a full length antibody under. Suchfusion results in two identical fusion peptides (either i) heavy chainand single chain Fab fragment or ii) light chain and single chain Fabfragment) which are coexpressed with either i) the light chain or theheavy chain of the full length antibody to give the bispecific antibodyaccording to the invention.

In a further embodiment said bispecific antibody is characterized inthat the constant region is of human origin.

In a further embodiment said bispecific antibody is characterized inthat the constant region of the bispecific antibody according to theinvention is of the human IgG1 subclass or of the human IgG1 subclasswith the mutations L234A and L235A.

In a further embodiment said bispecific antibody is characterized inthat the constant region of the bispecific antibody according to theinvention antibody is of the human IgG2 subclass.

In a further embodiment said bispecific antibody is characterized inthat the constant region of the bispecific antibody according to theinvention antibody is of the human IgG3 subclass.

In a further embodiment said bispecific antibody is characterized inthat the constant region of the bispecific antibody according to theinvention is of the human IgG4 subclass or of the human IgG4 subclasswith the additional mutation S228P.

It has now been found that the bispecific antibodies against human VEGFand human ANG-2 according to the current invention have improvedcharacteristics such as biological or pharmacological activity,pharmacokinetic properties or toxicity. They show increased in vivotumor growth inhibition and/or inhibition of tumor angiogenesis whencompared to the monospecific parent antibodies against VEGF and ANG-2(see Examples 16, 17 and 18: comparison of different bispecific<VEGF-ANG-2> antibodies bevacizumab-ANG2i-LC06 with the monospecificantibodies bevacicumab alone, ANG2i-LC06 alone, or both in combination).

Furthermore less toxic side effects (which is reflected in the improvedbody weight of the test animals as well as less deaths of test animalsduring the in vivo application) compared to the application of twocorresponding individual monospecific antibodies against VEGF and ANG-2in combination also represent an advantage of the bispecific antibodiesaccording to the invention.

Furthermore the bispecific antibodies according to the current inventionmay provide benefits such as reduced dose and/or frequency ofadministration and concomitantly cost savings.

The term “constant region” as used within the current applicationsdenotes the sum of the domains of an antibody other than the variableregion. The constant region is not involved directly in binding of anantigen, but exhibits various effector functions. Depending on the aminoacid sequence of the constant region of their heavy chains, antibodiesare divided in the classes: IgA, IgD, IgE, IgG and IgM, and several ofthese may be further divided into subclasses, such as IgG1, IgG2, IgG3,and IgG4, IgA1 and IgA2. The heavy chain constant regions thatcorrespond to the different classes of antibodies are called α, δ, ϵ, γ,and μ, respectively. The light chain constant regions which can be foundin all five antibody classes are called κ (kappa) and λ (lambda).

The term “constant region derived from human origin” as used in thecurrent application denotes a constant heavy chain region of a humanantibody of the subclass IgG1, IgG2, IgG3, or IgG4 and/or a constantlight chain kappa or lambda region. Such constant regions are well knownin the state of the art and e.g. described by Kabat, E. A., (see e.g.Johnson, G., and Wu, T. T., Nucleic Acids Res. 28 (2000) 214-218; Kabat,E. A., et al., Proc. Natl. Acad. Sci. USA 72 (1975) 2785-2788).

While antibodies of the IgG4 subclass show reduced Fc receptor(FcγRIIIa) binding, antibodies of other IgG subclasses show strongbinding. However Pro238, Asp265, Asp270, Asn297 (loss of Fccarbohydrate), Pro329, Leu234, Leu235, Gly236, Gly237, Ile253, Ser254,Lys288, Thr307, Gln311, Asn434, and His435 are residues which, ifaltered, provide also reduced Fc receptor binding (Shields, R. L., etal., J. Biol. Chem. 276 (2001) 6591-6604; Lund, J., et al., FASEB J. 9(1995) 115-119; Morgan, A., et al., Immunology 86 (1995) 319-324; EP 0307 434).

In one embodiment an antibody according to the invention has a reducedFcR binding compared to an IgG1 antibody and the monospecific bivalent(full length) parent antibody is in regard to FcR binding of IgG4subclass or of IgG1 or IgG2 subclass with a mutation in 5228, L234, L235and/or D265, and/or contains the PVA236 mutation. In one embodiment themutations in the monospecific bivalent (full length) parent antibody areS228P, L234A, L235A, L235E and/or PVA236. In another embodiment themutations in the monospecific bivalent (full length) parent antibody arein IgG4 S228P and in IgG1 L234A and L235A. Constant heavy chain regionsare shown in SEQ ID NO: 35 and 36. In one embodiment the constant heavychain region of the monospecific bivalent (full length) parent antibodyis of SEQ ID NO: 35 with mutations L234A and L235A. In anotherembodiment the constant heavy chain region of the monospecific bivalent(full length) parent antibody is of SEQ ID NO: 36 with mutation S228P.In another embodiment the constant light chain region of themonospecific bivalent (full length) parent antibody is a kappa lightchain region of SEQ ID NO: 37 or lambda light chain region of SEQ ID NO:34. Preferably the constant heavy chain region of the monospecificbivalent (full length) parent antibody is of SEQ ID NO: 35 or of SEQ IDNO: 36 with mutation S228P.

The constant region of an antibody is directly involved in ADCC(antibody-dependent cell-mediated cytotoxicity) and CDC(complement-dependent cytotoxicity). Complement activation (CDC) isinitiated by binding of complement factor C1q to the constant region ofmost IgG antibody subclasses. Binding of C1q to an antibody is caused bydefined protein-protein interactions at the so called binding site. Suchconstant region binding sites are known in the state of the art anddescribed e.g. by Lukas, T. J., et al., J. Immunol. 127 (1981)2555-2560; Brunhouse, R. and Cebra, J. J., Mol. Immunol. 16 (1979)907-917; Burton, D. R., et al., Nature 288 (1980) 338-344; Thommesen, J.E., et al., Mol. Immunol. 37 (2000) 995-1004; Idusogie, E. E., et al.,J. Immunol. 164 (2000) 4178-4184; Hezareh, M., et al., J. Virol. 75(2001) 12161-12168; Morgan, A., et al., Immunology 86 (1995) 319-324;and EP 0 307 434. Such constant region binding sites are, e.g.,characterized by the amino acids L234, L235, D270, N297, E318, K320,K322, P331, and P329 (numbering according to EU index of Kabat).

The term “antibody-dependent cellular cytotoxicity (ADCC)” refers tolysis of human target cells by an antibody according to the invention inthe presence of effector cells. ADCC is measured preferably by thetreatment of a preparation of CCR5 expressing cells with an antibodyaccording to the invention in the presence of effector cells such asfreshly isolated PBMC or purified effector cells from buffy coats, likemonocytes or natural killer (NK) cells or a permanently growing NK cellline.

The term “complement-dependent cytotoxicity (CDC)” denotes a processinitiated by binding of complement factor C1q to the Fc part of most IgGantibody subclasses. Binding of C1q to an antibody is caused by definedprotein-protein interactions at the so called binding site. Such Fc partbinding sites are known in the state of the art (see above). Such Fcpart binding sites are, e.g., characterized by the amino acids L234,L235, D270, N297, E318, K320, K322, P331, and P329 (numbering accordingto EU index of Kabat). Antibodies of subclass IgG1, IgG2, and IgG3usually show complement activation including C1q and C3 binding, whereasIgG4 does not activate the complement system and does not bind C1qand/or C3.

The antibody according to the invention is produced by recombinantmeans. Thus, one aspect of the current invention is a nucleic acidencoding the antibody according to the invention and a further aspect isa cell comprising said nucleic acid encoding an antibody according tothe invention. Methods for recombinant production are widely known inthe state of the art and comprise protein expression in prokaryotic andeukaryotic cells with subsequent isolation of the antibody and usuallypurification to a pharmaceutically acceptable purity. For the expressionof the antibodies as aforementioned in a host cell, nucleic acidsencoding the respective modified light and heavy chains are insertedinto expression vectors by standard methods. Expression is performed inappropriate prokaryotic or eukaryotic host cells like CHO cells, NS0cells, SP2/0 cells, HEK293 cells, COS cells, PER.C6 cells, yeast, or E.coli cells, and the antibody is recovered from the cells (supernatant orcells after lysis). General methods for recombinant production ofantibodies are well-known in the state of the art and described, forexample, in the review articles of Makrides, S. C., Protein Expr. Purif.17 (1999) 183-202; Geisse, S., et al., Protein Expr. Purif. 8 (1996)271-282; Kauffman, R. J., Mol. Biotechnol. 16 (2000) 151-161; Werner, R.G., Drug Res. 48 (1998) 870-880.

The bispecific antibodies are suitably separated from the culture mediumby conventional immunoglobulin purification procedures such as, forexample, protein A-Sepharose, hydroxylapatite chromatography, gelelectrophoresis, dialysis, or affinity chromatography. DNA and RNAencoding the monoclonal antibodies is readily isolated and sequencedusing conventional procedures. The hybridoma cells can serve as a sourceof such DNA and RNA. Once isolated, the DNA may be inserted intoexpression vectors, which are then transfected into host cells such asHEK 293 cells, CHO cells, or myeloma cells that do not otherwise produceimmunoglobulin protein, to obtain the synthesis of recombinantmonoclonal antibodies in the host cells.

Amino acid sequence variants (or mutants) of the bispecific antibody areprepared by introducing appropriate nucleotide changes into the antibodyDNA, or by nucleotide synthesis. Such modifications can be performed,however, only in a very limited range, e.g. as described above. Forexample, the modifications do not alter the above mentioned antibodycharacteristics such as the IgG isotype and antigen binding, but mayimprove the yield of the recombinant production, protein stability orfacilitate the purification.

The term “host cell” as used in the current application denotes any kindof cellular system which can be engineered to generate the antibodiesaccording to the current invention. In one embodiment HEK293 cells andCHO cells are used as host cells. As used herein, the expressions“cell,” “cell line,” and “cell culture” are used interchangeably and allsuch designations include progeny. Thus, the words “transformants” and“transformed cells” include the primary subject cell and culturesderived therefrom without regard for the number of transfers. It is alsounderstood that all progeny may not be precisely identical in DNAcontent, due to deliberate or inadvertent mutations. Variant progenythat have the same function or biological activity as screened for inthe originally transformed cell are included.

Expression in NS0 cells is described by, e.g., Barnes, L. M., et al.,Cytotechnology 32 (2000) 109-123; Barnes, L. M., et al., Biotech.Bioeng. 73 (2001) 261-270. Transient expression is described by, e.g.,Durocher, Y., et al., Nucl. Acids. Res. 30 (2002) E9. Cloning ofvariable domains is described by Orlandi, R., et al., Proc. Natl. Acad.Sci. USA 86 (1989) 3833-3837; Carter, P., et al., Proc. Natl. Acad. Sci.USA 89 (1992) 4285-4289; and Norderhaug, L., et al., J. Immunol. Methods204 (1997) 77-87. A preferred transient expression system (HEK 293) isdescribed by Schlaeger, E.-J., and Christensen, K., in Cytotechnology 30(1999) 71-83 and by Schlaeger, E.-J., in J. Immunol. Methods 194 (1996)191-199.

The control sequences that are suitable for prokaryotes, for example,include a promoter, optionally an operator sequence, and a ribosomebinding site. Eukaryotic cells are known to utilize promoters, enhancersand polyadenylation signals.

A nucleic acid is “operably linked” when it is placed in a functionalrelationship with another nucleic acid sequence. For example, DNA for apre-sequence or secretory leader is operably linked to DNA for apolypeptide if it is expressed as a pre-protein that participates in thesecretion of the polypeptide; a promoter or enhancer is operably linkedto a coding sequence if it affects the transcription of the sequence; ora ribosome binding site is operably linked to a coding sequence if it ispositioned so as to facilitate translation. Generally, “operably linked”means that the DNA sequences being linked are contiguous, and, in thecase of a secretory leader, contiguous and in reading frame. However,enhancers do not have to be contiguous. Linking is accomplished byligation at convenient restriction sites. If such sites do not exist,the synthetic oligonucleotide adaptors or linkers are used in accordancewith conventional practice.

Purification of antibodies is performed in order to eliminate cellularcomponents or other contaminants, e.g. other cellular nucleic acids orproteins, by standard techniques, including alkaline/SDS treatment, CsClbanding, column chromatography, agarose gel electrophoresis, and otherswell known in the art. See Ausubel, F., et al., ed. Current Protocols inMolecular Biology, Greene Publishing and Wiley Interscience, New York(1987). Different methods are well established and widely used forprotein purification, such as affinity chromatography with microbialproteins (e.g. protein A or protein G affinity chromatography), ionexchange chromatography (e.g. cation exchange (carboxymethyl resins),anion exchange (amino ethyl resins) and mixed-mode exchange), thiophilicadsorption (e.g. with beta-mercaptoethanol and other SH ligands),hydrophobic interaction or aromatic adsorption chromatography (e.g. withphenyl-sepharose, aza-arenophilic resins, or m-aminophenylboronic acid),metal chelate affinity chromatography (e.g. with Ni(II)- andCu(II)-affinity material), size exclusion chromatography, andelectrophoretical methods (such as gel electrophoresis, capillaryelectrophoresis) (Vijayalakshmi, M. A., Appl. Biochem. Biotech. 75(1998) 93-102).

One aspect of the invention is a pharmaceutical composition comprisingan antibody according to the invention. Another aspect of the inventionis the use of an antibody according to the invention for the manufactureof a pharmaceutical composition. A further aspect of the invention is amethod for the manufacture of a pharmaceutical composition comprising anantibody according to the invention. In another aspect, the presentinvention provides a composition, e.g. a pharmaceutical composition,containing an antibody according to the present invention, formulatedtogether with a pharmaceutical carrier.

One embodiment of the invention is the bispecific antibody according tothe invention for the treatment of cancer.

Another aspect of the invention is said pharmaceutical composition forthe treatment of cancer.

Another aspect of the invention is the use of an antibody according tothe invention for the manufacture of a medicament for the treatment ofcancer.

Another aspect of the invention is method of treatment of a patientsuffering from cancer comprising the step of administering an antibodyaccording to the invention to a patient in the need of such treatment.

As used herein, “pharmaceutical carrier” includes any and all solvents,dispersion media, coatings, antibacterial and antifungal agents,isotonic and absorption delaying agents, and the like that arephysiologically compatible. Preferably, the carrier is suitable forintravenous, intramuscular, subcutaneous, parenteral, spinal orepidermal administration (e.g. by injection or infusion).

A composition of the present invention can be administered by a varietyof methods known in the art. As will be appreciated by the skilledartisan, the route and/or mode of administration will vary dependingupon the desired results. To administer a compound of the invention bycertain routes of administration, it may be necessary to coat thecompound with, or co-administer the compound with, a material to preventits inactivation. For example, the compound may be administered to asubject in an appropriate carrier, for example, liposomes, or a diluent.Pharmaceutically acceptable diluents include saline and aqueous buffersolutions. Pharmaceutical carriers include sterile aqueous solutions ordispersions and sterile powders for the extemporaneous preparation ofsterile injectable solutions or dispersion. The use of such media andagents for pharmaceutically active substances is known in the art.

The phrases “parenteral administration” and “administered parenterally”as used herein means modes of administration other than enteral andtopical administration, usually by injection, and includes, withoutlimitation, intravenous, intramuscular, intra-arterial, intrathecal,intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal,transtracheal, subcutaneous, subcuticular, intra-articular, subcapsular,subarachnoid, intraspinal, epidural and intrasternal injection andinfusion.

The term cancer as used herein refers to proliferative diseases, such aslymphomas, lymphocytic leukemias, lung cancer, non small cell lung(NSCL) cancer, bronchioloalviolar cell lung cancer, bone cancer,pancreatic cancer, skin cancer, cancer of the head or neck, cutaneous orintraocular melanoma, uterine cancer, ovarian cancer, rectal cancer,cancer of the anal region, stomach cancer, gastric cancer, colon cancer,breast cancer, uterine cancer, carcinoma of the fallopian tubes,carcinoma of the endometrium, carcinoma of the cervix, carcinoma of thevagina, carcinoma of the vulva, Hodgkin's Disease, cancer of theesophagus, cancer of the small intestine, cancer of the endocrinesystem, cancer of the thyroid gland, cancer of the parathyroid gland,cancer of the adrenal gland, sarcoma of soft tissue, cancer of theurethra, cancer of the penis, prostate cancer, cancer of the bladder,cancer of the kidney or ureter, renal cell carcinoma, carcinoma of therenal pelvis, mesothelioma, hepatocellular cancer, biliary cancer,neoplasms of the central nervous system (CNS), spinal axis tumors, brainstem glioma, glioblastoma multiforme, astrocytomas, schwanomas,ependymonas, medulloblastomas, meningiomas, squamous cell carcinomas,pituitary adenoma and Ewings sarcoma, including refractory versions ofany of the above cancers, or a combination of one or more of the abovecancers.

Another aspect of the invention is the bispecific antibody according tothe invention or said pharmaceutical composition as anti-angiogenicagent. Such anti-angiogenic agent can be used for the treatment ofcancer, especially solid tumors, and other vascular diseases.

One embodiment of the invention is the bispecific antibody according tothe invention for the treatment of vascular diseases.

Another aspect of the invention is said pharmaceutical composition forthe treatment of vascular diseases.

Another aspect of the invention is the use of an antibody according tothe invention for the manufacture of a medicament for the treatment ofvascular diseases.

Another aspect of the invention is method of treatment of a patientsuffering from a vascular disease comprising the step of administeringan antibody according to the invention to a patient in the need of suchtreatment.

The term “vascular disease” includes Cancer, Inflammatory diseases,Atherosclerosis, Ischemia, Trauma, Sepsis, COPD, Asthma, Diabetes, AMD,Retinopathy, Stroke, Adipositas, Acute lung injury, Hemorrhage, Vascularleak e.g. Cytokine induced, Allergy, Graves' Disease, Hashimoto'sAutoimmune Thyroiditis, Idiopathic Thrombocytopenic Purpura, Giant CellArteritis, Rheumatoid Arthritis, Systemic Lupus Erythematosus (SLE),Lupus Nephritis, Crohn's Disease, Multiple Sclerosis, UlcerativeColitis, especially to solid tumors, intraocular neovascular syndromessuch as proliferative retinopathies or age-related macular degeneration(AMD), rheumatoid arthritis, and psoriasis (Folkman, J., et al., J.Biol. Chem. 267 (1992) 10931-10934; Klagsbrun, M., et al., Annu. Rev.Physiol. 53 (1991) 217-239; and Garner, A., Vascular diseases, In:Pathobiology of ocular disease, A dynamic approach, Garner, A., andKlintworth, G. K., (eds.), 2nd edition, Marcel Dekker, New York (1994),pp 1625-1710).

These compositions may also contain adjuvants such as preservatives,wetting agents, emulsifying agents and dispersing agents. Prevention ofpresence of microorganisms may be ensured both by sterilizationprocedures, supra, and by the inclusion of various antibacterial andantifungal agents, for example, paraben, chlorobutanol, phenol, sorbicacid, and the like. It may also be desirable to include isotonic agents,such as sugars, sodium chloride, and the like into the compositions. Inaddition, prolonged absorption of the injectable pharmaceutical form maybe brought about by the inclusion of agents which delay absorption suchas aluminum monostearate and gelatin.

Regardless of the route of administration selected, the compounds of thepresent invention, which may be used in a suitable hydrated form, and/orthe pharmaceutical compositions of the present invention, are formulatedinto pharmaceutically acceptable dosage forms by conventional methodsknown to those of skill in the art.

Actual dosage levels of the active ingredients in the pharmaceuticalcompositions of the present invention may be varied so as to obtain anamount of the active ingredient which is effective to achieve thedesired therapeutic response for a particular patient, composition, andmode of administration, without being toxic to the patient. The selecteddosage level will depend upon a variety of pharmacokinetic factorsincluding the activity of the particular compositions of the presentinvention employed, the route of administration, the time ofadministration, the rate of excretion of the particular compound beingemployed, the duration of the treatment, other drugs, compounds and/ormaterials used in combination with the particular compositions employed,the age, sex, weight, condition, general health and prior medicalhistory of the patient being treated, and like factors well known in themedical arts.

The composition must be sterile and fluid to the extent that thecomposition is deliverable by syringe. In addition to water, the carrierpreferably is an isotonic buffered saline solution.

Proper fluidity can be maintained, for example, by use of a coating suchas lecithin, by maintenance of required particle size in the case ofdispersion, or by use of surfactants. In many cases, it is preferable toinclude isotonic agents, for example, sugars, polyalcohols such asmannitol or sorbitol, and sodium chloride in the composition.

As used herein, the expressions “cell,” “cell line,” and “cell culture”are used interchangeably and all such designations include progeny.Thus, the words “transformants” and “transformed cells” include theprimary subject cell and cultures derived therefrom without regard forthe number of transfers. It is also understood that all progeny may notbe precisely identical in DNA content, due to deliberate or inadvertentmutations. Variant progeny that have the same function or biologicalactivity as screened for in the originally transformed cell areincluded. Where distinct designations are intended, it will be clearfrom the context.

The term “transformation” as used herein refers to process of transferof a vectors/nucleic acid into a host cell. If cells without formidablecell wall barriers are used as host cells, transfection is carried oute.g. by the calcium phosphate precipitation method as described byGraham, F. L., van der Eb, A. J., Virology 52 (1978) 546ff. However,other methods for introducing DNA into cells such as by nuclearinjection or by protoplast fusion may also be used. If prokaryotic cellsor cells which contain substantial cell wall constructions are used,e.g. one method of transfection is calcium treatment using calciumchloride as described by Cohen, S. N., et al., PNAS. 69 (1972)2110-2114.

As used herein, “expression” refers to the process by which a nucleicacid is transcribed into mRNA and/or to the process by which thetranscribed mRNA (also referred to as transcript) is subsequently beingtranslated into peptides, polypeptides, or proteins. The transcripts andthe encoded polypeptides are collectively referred to as gene product.If the polynucleotide is derived from genomic DNA, expression in aeukaryotic cell may include splicing of the mRNA.

A “vector” is a nucleic acid molecule, in particular self-replicating,which transfers an inserted nucleic acid molecule into and/or betweenhost cells. The term includes vectors that function primarily forinsertion of DNA or RNA into a cell (e.g., chromosomal integration),replication of vectors that function primarily for the replication ofDNA or RNA, and expression vectors that function for transcriptionand/or translation of the DNA or RNA. Also included are vectors thatprovide more than one of the functions as described.

An “expression vector” is a polynucleotide which, when introduced intoan appropriate host cell, can be transcribed and translated into apolypeptide. An “expression system” usually refers to a suitable hostcell comprised of an expression vector that can function to yield adesired expression product.

EXAMPLES

The following examples, and aforementioned sequence listing and figuresare provided to aid the understanding of the present invention, the truescope of which is set forth in the appended claims. It is understoodthat modifications can be made in the procedures set forth withoutdeparting from the spirit of the invention.

Materials & General Methods

General information regarding the nucleotide sequences of humanimmunoglobulins light and heavy chains is given in: Kabat, E. A., etal., Sequences of Proteins of Immunological Interest, 5th ed., PublicHealth Service, National Institutes of Health, Bethesda, Md. (1991).Amino acids of antibody chains are numbered and referred to according toEU numbering (Edelman, G. M., et al., Proc. Natl. Acad. Sci. USA 63(1969) 78-85; Kabat, E. A., et al., Sequences of Proteins ofImmunological Interest, 5th ed., Public Health Service, NationalInstitutes of Health, Bethesda, Md., (1991)).

Recombinant DNA Techniques

Standard methods were used to manipulate DNA as described in Sambrook,J. et al., Molecular cloning: A laboratory manual; Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y., 1989. The molecularbiological reagents were used according to the manufacturer'sinstructions.

Gene Synthesis

Desired gene segments were prepared from oligonucleotides made bychemical synthesis. The gene segments, which are flanked by singularrestriction endonuclease cleavage sites, were assembled by annealing andligation of oligonucleotides including PCR amplification andsubsequently cloned via the indicated restriction sites e.g. KpnI/SacIor AscI/PacI into a pPCRScript (Stratagene) based pGA4 cloning vector.The DNA sequences of the subcloned gene fragments were confirmed by DNAsequencing. Gene synthesis fragments were ordered according to givenspecifications at Geneart (Regensburg, Germany). All gene segmentsencoding light and heavy chains of Ang-2/VEGF bispecific antibodies weresynthesized with a 5′-end DNA sequence coding for a leader peptide(MGWSCIILFLVATATGVHS), which targets proteins for secretion ineukaryotic cells, and 5′-BamHI and 3′-XbaI restriction sites. DNAsequences carrying disulfide stabilized “knobs-into-hole” modified heavychains were designed with S354C and T366W mutations in the “knobs” heavychain and Y349C, T366S, L368A and Y407V mutations in the “hole” heavychain.

DNA Sequence Determination

DNA sequences were determined by double strand sequencing performed atMediGenomix GmbH (Martinsried, Germany) or Sequiserve GmbH(Vaterstetten, Germany).

DNA and Protein Sequence Analysis and Sequence Data Management

The GCG's (Genetics Computer Group, Madison, Wis.) software packageversion 10.2 and Infomax's Vector NT1 Advance suite version 8.0 was usedfor sequence creation, mapping, analysis, annotation and illustration.

Expression Vectors (for Example 1)

For the expression of the described antibodies, variants of expressionplasmids for transient expression (e.g. in HEK293 EBNA or HEK293-F)cells or for stable expression (e.g. in CHO cells) based either on acDNA organization with a CMV-Intron A promoter or on a genomicorganization with a CMV promoter (e.g. FIG. 2B) were applied.

Beside the antibody expression cassette the vectors contained:

-   -   an origin of replication which allows replication of this        plasmid in E. coli, and    -   a ß-lactamase gene which confers ampicillin resistance in E.        coli.

The transcription unit of the antibody gene is composed of the followingelements:

-   -   unique restriction site(s) at the 5′ end    -   the immediate early enhancer and promoter from the human        cytomegalovirus,    -   followed by the Intron A sequence in the case of the cDNA        organization,    -   a 5′-untranslated region of a human antibody gene,    -   a immunoglobulin heavy chain signal sequence,    -   the human antibody chain (heavy chain, modified heavy chain or        light chain) either as cDNA or as genomic organization with an        the immunoglobulin exon-intron organization    -   a 3′ untranslated region with a polyadenylation signal sequence,        and    -   unique restriction site(s) at the 3′ end.

The fusion genes comprising the heavy chain sequences of the selectedantibody and the C-terminal scFv fusion as described below weregenerated by PCR and/or gene synthesis and assembled with knownrecombinant methods and techniques by connection of the nucleic acidsegments, for example, using unique NsiI and EcoRI sites in the genomicheavy chain vectors. The subcloned nucleic acid sequences were verifiedby DNA sequencing. For transient and stable transfections largerquantities of the plasmids were prepared by plasmid preparation fromtransformed E. coli cultures (Nucleobond AX, Macherey-Nagel).

Expression Vectors (for Example 10-14)

An expression vector was used which composed of the following elements:

-   -   a hygromycin resistance gene as a selection marker,    -   an origin of replication, oriP, of Epstein-Barr virus (EBV),    -   an origin of replication from the vector pUC18 which allows        replication of this plasmid in E. coli    -   a beta-lactamase gene which confers ampicillin resistance in E.        coli,    -   the immediate early enhancer and promoter from the human        cytomegalovirus (HCMV),    -   the human 1-immunoglobulin polyadenylation (“poly A”) signal        sequence, and    -   unique BamHI and XbaI restriction sites.

The immunoglobulin fusion genes comprising the heavy or light chainconstructs as well as “knobs-into-hole” constructs with C-terminal VHand VL domains were prepared by gene synthesis and cloned into pGA18(ampR) plasmids as described. The pG18 (ampR) plasmids carrying thesynthesized DNA segments and the Roche expression vector were digestedwith BamHI and XbaI restriction enzymes (Roche Molecular Biochemicals)and subjected to agarose gel electrophoresis. Purified heavy and lightchain coding DNA segments were then ligated to the isolated Rocheexpression vector BamHI/XbaI fragment resulting in the final expressionvectors. The final expression vectors were transformed into E. colicells, expression plasmid DNA was isolated (Miniprep) and subjected torestriction enzyme analysis and DNA sequencing. Correct clones weregrown in 150 ml LB-Amp medium, again plasmid DNA was isolated (Maxiprep)and sequence integrity confirmed by DNA sequencing.

Cell Culture Techniques

Standard cell culture techniques were used as described in CurrentProtocols in Cell Biology (2000), Bonifacino, J. S., Dasso, M., Harford,J. B., Lippincott-Schwartz, J. and Yamada, K. M. (eds.), John Wiley &Sons, Inc.

Transient Transfections in HEK293-F System (for Example 1)

Antibodies were generated by transient transfection of the two plasmidsencoding the heavy or modified heavy chain, respectively and thecorresponding light chain using the HEK293-F system (Invitrogen)according to the manufacturer's instruction. Briefly, HEK293-F cells(Invitrogen) growing in suspension either in a shake flask or in astirred fermenter in serumfree FreeStyle 293 expression medium(Invitrogen) were transfected with a mix of the two respectiveexpression plasmids and 293fectin or fectin (Invitrogen). For e.g. 2 Lshake flask (Corning) HEK293-F cells were seeded at a density of 1.0E*6cells/mL in 600 mL and incubated at 120 rpm, 8% CO2. The day after thecells were transfected at a cell density of ca. 1.5E*6 cells/mL with ca.42 mL mix of A) 20 mL Opti-MEM (Invitrogen) with 600 μg total plasmidDNA (1 μg/mL) encoding the heavy or modified heavy chain, respectivelyand the corresponding light chain in an equimolar ratio and B) 20 mlOpti-MEM+1.2 mL 293 fectin or fectin (2 μl/mL). According to the glucoseconsumption glucose solution was added during the course of thefermentation. The supernatant containing the secreted antibody washarvested after 5-10 days and antibodies were either directly purifiedfrom the supernatant or the supernatant was frozen and stored.

Transient Transfections in HEK293-F System (for Example 10-14)

Recombinant immunoglobulin variants were expressed by transienttransfection of human embryonic kidney 293-F cells using the FreeStyle™293 Expression System according to the manufacturer's instruction(Invitrogen, USA). Briefly, suspension FreeStyle™ 293-F cells werecultivated in FreeStyle™ 293 Expression medium at 37° C./8% CO₂ and thecells were seeded in fresh medium at a density of 1-2×10⁶ viablecells/ml on the day of transfection. DNA-293Fectin™ complexes wereprepared in Opti-MEM® I medium (Invitrogen, USA) using 325 μl of293Fectin™ (Invitrogen, Germany) and 250 μg of heavy and light chainplasmid DNA in a 1:1 molar ratio for a 250 ml final transfection volume.“Knobs-into-hole” DNA-293fectin complexes with two heavy chains and onelight chain were prepared in Opti-MEM® I medium (Invitrogen, USA) using325 μl of 293Fectin™ (Invitrogen, Germany) and 250 μg of“Knobs-into-hole” heavy chain 1 and 2 and light chain plasmid DNA in a1:1:2 molar ratio for a 250 ml final transfection volume.“Knobs-into-hole” DNA-293fectin complexes with two heavy chains wereprepared in Opti-MEM® I medium (Invitrogen, USA) using 325 μl of293Fectin™ (Invitrogen, Germany) and 250 μg of “Knobs-into-hole” heavychain 1 and 2 DNA in a 1:1 molar ratio for a 250 ml final transfectionvolume. CrossMab DNA-293fectin complexes were prepared in Opti-MEM® Imedium (Invitrogen, USA) using 325 μl of 293Fectin™ (Invitrogen,Germany) and 250 of “Knobs-into-hole” heavy chain 1 and 2 and lightchain plasmid DNA in a 1:1:1:1 molar ratio for a 250 ml finaltransfection volume. Antibody containing cell culture supernatants wereharvested 7 days after transfection by centrifugation at 14000 g for 30minutes and filtered through a sterile filter (0.22 μm). Supernatantswere stored at −20° C. until purification.

Protein Determination

The protein concentration of purified antibodies and derivatives wasdetermined by determining the optical density (OD) at 280 nm, using themolar extinction coefficient calculated on the basis of the amino acidsequence according to Pace et. al., Protein Science, 1995, 4, 2411-1423.

Antibody Concentration Determination in Supernatants

The concentration of antibodies and derivatives in cell culturesupernatants was estimated by immunoprecipitation with Protein AAgarose-beads (Roche). 60 μL Protein A Agarose beads are washed threetimes in TBS-NP40 (50 mM Tris, pH 7.5, 150 mM NaCl, 1% Nonidet-P40).Subsequently, 1-15 mL cell culture supernatant are applied to theProtein A Agarose beads pre-equilibrated in TBS-NP40. After incubationfor at 1 h at room temperature the beads are washed on anUltrafree-MC-filter column (Amicon] once with 0.5 mL TBS-NP40, twicewith 0.5 mL 2× phosphate buffered saline (2×PBS, Roche) and briefly fourtimes with 0.5 mL 100 mM Na-citrate pH 5.0. Bound antibody is eluted byaddition of 35 μl NuPAGE® LDS Sample Buffer (Invitrogen). Half of thesample is combined with NuPAGE® Sample Reducing Agent or left unreduced,respectively, and heated for 10 min at 70° C. Consequently, 20 μl areapplied to an 4-12% NuPAGE® Bis-Tris SDS-PAGE (Invitrogen) (with MOPSbuffer for non-reduced SDS-PAGE and MES buffer with NuPAGE® Antioxidantrunning buffer additive (Invitrogen) for reduced SDS-PAGE) and stainedwith Coomassie Blue.

The concentration of antibodies and derivatives in cell culturesupernatants was measured by Protein A-HPLC chromatography. Briefly,cell culture supernatants containing antibodies and derivatives thatbind to Protein A were applied to a HiTrap Protein A column (GEHealthcare) in 50 mM K2HPO4, 300 mM NaCl, pH 7.3 and eluted from thematrix with 550 mM acetic acid, pH 2.5 on a Dionex HPLC-System. Theeluted protein was quantified by UV absorbance and integration of peakareas. A purified standard IgG1 antibody served as a standard.

Alternatively, the concentration of antibodies and derivatives in cellculture supernatants was measured by Sandwich-IgG-ELISA. Briefly,StreptaWell High Bind Strepatavidin A-96 well microtiter plates (Roche)were coated with 100 μL/well biotinylated anti-human IgG capturemolecule F(ab′)2<h-Fcgamma> BI (Dianova) at 0.1 μg/mL for 1 h at roomtemperature or alternatively over night at 4° C. and subsequently washedthree times with 200 μL/well PBS, 0.05% Tween (PBST, Sigma). 100 μL/wellof a dilution series in PBS (Sigma) of the respective antibodycontaining cell culture supernatants was added to the wells andincubated for 1-2 h on a microtiterplate shaker at room temperature. Thewells were washed three times with 200 μL/well PBST and bound antibodywas detected with 100 μl F(ab′)2<hFcgamma>POD (Dianova) at 0.1 μg/mL asdetection antibody for 1-2 h on a microtiterplate shaker at roomtemperature. Unbound detection antibody was washed away three times with200 μL/well PBST and the bound detection antibody was detected byaddition of 100 μL ABTS/well. Determination of absorbance was performedon a Tecan Fluor Spectrometer at a measurement wavelength of 405 nm(reference wavelength 492 nm).

Protein Purification

Proteins were purified from filtered cell culture supernatants referringto standard protocols. In brief, antibodies were applied to a Protein ASepharose column (GE Healthcare) and washed with PBS. Elution ofantibodies was achieved at acidic pH followed by immediateneutralization of the sample. Aggregated protein was separated frommonomeric antibodies by size exclusion chromatography (Superdex 200, GEHealthcare) in 20 mM Histidine, 140 mM NaCl pH 6.0. Monomeric antibodyfractions were pooled, concentrated if required using e.g. a MILLIPOREAmicon Ultra (30 MWCO) centrifugal concentrator and stored at −80° C.Part of the samples were provided for subsequent protein analytics andanalytical characterization e.g. by SDS-PAGE, size exclusionchromatography, mass spectrometry and Endotoxin determination (see FIGS.3 and 4).

SDS-PAGE

The NuPAGE® Pre-Cast gel system (Invitrogen) was used according to themanufacturer's instruction. In particular, 4-20% NuPAGE® Novex®TRIS-Glycine Pre-Cast gels and a Novex® TRIS-Glycine SDS running bufferwere used. (see e.g. FIG. 3). Reducing of samples was achieved by addingNuPAGE® sample reducing agent prior to running the gel.

Analytical Size Exclusion Chromatography

Size exclusion chromatography for the determination of the aggregationand oligomeric state of antibodies was performed by HPLC chromatography.Briefly, Protein A purified antibodies were applied to a Tosoh TSKgelG3000SW column in 300 mM NaCl, 50 mM KH2PO4/K2HPO4, pH 7.5 on an AgilentHPLC 1100 system or to a Superdex 200 column (GE Healthcare) in 2×PBS ona Dionex HPLC-System. The eluted protein was quantified by UV absorbanceand integration of peak areas. BioRad Gel Filtration Standard 151-1901served as a standard. (see e.g. FIG. 4A-C).

Mass Spectrometry

The total deglycosylated mass of crossover antibodies was determined andconfirmed via electrospray ionization mass spectrometry (ESI-MS).Briefly, 100 μg purified antibodies were deglycosylated with 50 mUN-Glycosidase F (PNGaseF, ProZyme) in 100 mM KH2PO4/K2HPO4, pH 7 at 37°C. for 12-24 h at a protein concentration of up to 2 mg/ml andsubsequently desalted via HPLC on a Sephadex G25 column (GE Healthcare).The mass of the respective heavy and light chains was determined byESI-MS after deglycosylation and reduction. In brief, 50 μg antibody in115 μl were incubated with 60 μl 1M TCEP and 50 μl 8 MGuanidine-hydrochloride subsequently desalted. The total mass and themass of the reduced heavy and light chains was determined via ESI-MS ona Q-Star Elite MS system equipped with a NanoMate source.

VEGF Binding ELISA

The binding properties of the tetravalent antibodies (TvAb) wasevaluated in an ELISA assay with full-length VEGF165-His protein (R&DSystems) (FIG. 5). For this sake Falcon polystyrene clear enhancedmicrotiter plates were coated with 100 μl 2 μg/mL recombinant humanVEGF165 (R&D Systems) in PBS for 2 h at room temperature or over nightat 4° C. The wells were washed three times with 300 μl PBST (0.2% Tween20) and blocked with 200 μl 2% BSA 0.1% Tween 20 for 30 min at roomtemperature and subsequently washed three times with 300 μl PBST. 100μL/well of a dilution series (40 pM-0.01 pM) of purified <VEGF-ANG-2>TvAb and as a reference the human anti-ANG-2 antibody <ANG-2> antibodyMab536 (Oliner et al., Cancer Cell. 2004 November; 6(5):507-16, US2006/0122370) and the anti VEGF antibody <VEGF> antibody G6-31 (Liang etal., J Biol Chem. 2006 Jan. 13; 281(2):951-61; US 2007/0141065) in PBS(Sigma) was added to the wells and incubated for 1 h on amicrotiterplate shaker at room temperature. The wells were washed threetimes with 300 μl PBST (0.2% Tween 20) and bound antibody was detectedwith 100 μL/well 0.1 μg/ml F(ab′) <hFcgamma>POD (Immuno research) in 2%BSA 0.1% Tween 20 as detection antibody for 1 h on a microtiterplateshaker at room temperature. Unbound detection antibody was washed awaythree times with 300 μL/well PBST and the bound detection antibody wasdetected by addition of 100 μL ABTS/well. Determination of absorbancewas performed on a Tecan Fluor Spectrometer at a measurement wavelengthof 405 nm (reference wavelength 492 nm).

VEGF Binding: Kinetic Characterization of VEGF Binding at 37° C. bySurface Plasmon Resonance (Biacore)

In order to further corroborate the ELISA findings the binding of <VEGF>antibodies G6-31 or bevacizumab and <VEGF-Ang-2> TvAb6 orTvAb-2441-bevacizumab-LC06 or TvAb-2441-bevacizumab-LC08 to VEGF wasquantitatively analyzed using surface plasmon resonance technology on aBiacore T100 instrument according to the following protocol and analyzedusing the T100 software package: Briefly <VEGF> antibodies were capturedon a CM5-Chip via binding to a Goat Anti Human IgG (JIR 109-005-098).The capture antibody was immobilized by amino coupling using standardamino coupling as follows: HBS-N buffer served as running buffer,activation was done by mixture of EDC/NHS with the aim for a liganddensity of 700 RU. The Capture-Antibody was diluted in coupling bufferNaAc, pH 5.0, c=2 μg/mL, finally still activated carboxyl groups wereblocked by injection of 1 M Ethanolamine. Capturing of Mabs <VEGF>antibodies was done at a flow of 5 μL/min and c(Mabs<VEGF>)=10 nM,diluted with running buffer+1 mg/mL BSA; a capture level of approx. 30RU should be reached. rhVEGF (rhVEGF, R&D-Systems Cat.-No, 293-VE) wasused as analyte. The kinetic characterization of VEGF binding to <VEGF>antibodies was performed at 37° C. in PBS+0.005% (v/v) Tween20 asrunning buffer. The sample was injected with a flow of 50 μL/min and anassociation of time 80 sec. and a dissociation time of 1200 sec with aconcentration series of rhVEGF from 300-0.29 nM. Regeneration of freecapture antibody surface was performed with 10 mM Glycin pH 1.5 and acontact time of 60 sec after each analyte cycle. Kinetic constants werecalculated by using the usual double referencing method (controlreference: binding of rhVEGF to capture molecule Goat Anti Human IgG,blanks on the measuring flow cell, rhVEGF concentration “0”, Model:Langmuir binding 1:1, (Rmax set to local because of capture moleculebinding). FIG. 11 shows a schematic view of the Biacore assay.

ANG-2 Binding ELISA

The binding properties of the tetravalent antibodies (TvAb) wasevaluated in an ELISA assay with full-length Angiopoietin-2-His protein(R&D Systems) (FIG. 6a ). For this sake Falcon polystyrene clearenhanced microtiter plates were coated with 100 μl μg/mL recombinanthuman Angiopoietin-2 (R&D Systems, carrier-free) in PBS for 2 h at roomtemperature or over night at 4° C. The wells were washed three timeswith 300 μl PBST (0.2% Tween 20) and blocked with 200 μl 2% BSA 0.1%Tween 20 for 30 min at room temperature and subsequently washed threetimes with 300 μl PBST. 100 μL/well of a dilution series (40 pM-0.01 pM)of purified <VEGF-ANG-2> TvAb and as a reference <ANG-2> antibody Mab536and VEGF> antibody G6-31 in PBS (Sigma) was added to the wells andincubated for 1 h on a microtiterplate shaker at room temperature. Thewells were washed three times with 300 μl PBST (0.2% Tween 20) and boundantibody was detected with 100 μL/well 0.1 μg/ml F(ab′) <hk>POD (BiozolCat. No. 206005) in 2% BSA 0.1% Tween 20 as detection antibody for 1 hon a microtiterplate shaker at room temperature. Unbound detectionantibody was washed away three times with 300 μL/well PBST and the bounddetection antibody was detected by addition of 100 μL ABTS/well.Determination of absorbance was performed on a Tecan Fluor Spectrometerat a measurement wavelength of 405 nm (reference wavelength 492 nm).

Comparative Binding to ANG-1 and ANG-2 (ANG-1 and ANG-2 Binding ELISA)

The binding properties of antibodies were evaluated in an ELISA assaywith full-length Angiopoietin-2-His protein (R&D Systems #623-AN/CF orin house produced material) or Angiopoietin-1-His (R&D systems #923-AN).Therefore 96 well plates (Falcon polystyrene clear enhanced microtiterplates or Nunc Maxisorb) were coated with 100 μl 1 μg/mL recombinanthuman Angiopoietin-1 or Angiopoietin-2 (carrier-free) in PBS (Sigma) for2 h at room temperature or over night at 4° C. The wells were washedthree times with 300 μl PBST (0.2% Tween 20) and blocked with 200 μl 2%BSA 0.1% Tween 20 for 30 min at room temperature and subsequently washedthree times with 300 μl PBST. 100 μL/well of a dilution series (40pM-0.01 pM) of purified test antibody in PBS was added to the wells andincubated for 1 h on a microtiterplate shaker at room temperature. Thewells were washed three times with 300 μl PBST (0.2% Tween 20) and boundantibody was detected with 100 μL/well 0.1 μg/ml F(ab′)<hk>POD (BiozolCat. No. 206005) in 2% BSA 0.1% Tween 20 as detection antibody for 1 hon a microtiterplate shaker at room temperature. Unbound detectionantibody was washed away three times with 300 μL/well PBST and the bounddetection antibody was detected by addition of 100 μL ABTS/well.Determination of absorbance was performed on a Tecan Fluor Spectrometerat a measurement wavelength of 405 nm (reference wavelength 492 nm).

ANG-2 Binding BIACORE

Binding of the antibodies to the antigen e.g. human ANG-2 wereinvestigated by surface plasmon resonance using a BIACORE T100instrument (GE Healthcare Biosciences AB, Uppsala, Sweden). Briefly, foraffinity measurements goat<hIgG-Fcgamma> polyclonal antibodies wereimmobilized on a CM5 chip via amine coupling for presentation of theantibodies against human ANG-2 (FIG. 6B). Binding was measured in HBSbuffer (HBS-P (10 mM HEPES, 150 mM NaCl, 0.005% Tween 20, ph 7.4), 25°C. Purified ANG-2-His (R&D systems or in house purified) was added invarious concentrations between 6.25 nM and 200 nM in solution.Association was measured by an ANG-2-injection of 3 minutes;dissociation was measured by washing the chip surface with HBS bufferfor 3 minutes and a K_(D) value was estimated using a 1:1 Langmuirbinding model. Due to heterogenity of the ANG-2 preparation no 1:1binding could be observed; K_(D) values are thus only relativeestimations. Negative control data (e.g. buffer curves) were subtractedfrom sample curves for correction of system intrinsic baseline drift andfor noise signal reduction. Biacore T100 Evaluation Software version1.1.1 was used for analysis of sensorgrams and for calculation ofaffinity data. Alternatively, Ang-2 could be captured with a capturelevel of 2000-1700 RU via a PentaHisAntibody (PentaHis-Ab BSA-free,Qiagen No. 34660) that was immobilized on a CMS chip via amine coupling(BSA-free) (see below).

Inhibition of huANG-2 binding to Tie-2 (ELISA)

The interaction ELISA was performed on 384 well microtiter plates(MicroCoat, DE, Cat. No. 464718) at RT. After each incubation stepplates were washed 3 times with PBST. ELISA plates were coated with 0.5μg/ml Tie-2 protein (R&D Systems, UK, Cat. No. 313-TI) for at least 2hours (h). Thereafter the wells were blocked with PBS supplemented with0.2% Tween-20 and 2% BSA (Roche Diagnostics GmbH, DE) for 1 h. Dilutionsof purified antibodies in PBS were incubated together with 0.2 μg/mlhuAngiopoietin-2 (R&D Systems, UK, Cat. No. 623-AN) for 1 h at RT. Afterwashing a mixture of 0.5 μg/ml biotinylated anti-Angiopoietin-2 cloneBAM0981 (R&D Systems, UK) and 1:3000 diluted streptavidin HRP (RocheDiagnostics GmbH, DE, Cat. No. 11089153001) was added for 1 h.Thereafter the plates were washed 6 times with PBST. Plates weredeveloped with freshly prepared ABTS reagent (Roche Diagnostics GmbH,DE, buffer #204 530 001, tablets #11 112 422 001) for 30 minutes at RT.Absorbance was measured at 405 nm.

ANG-2-VEGF Bridging ELISA

The binding properties of the tetravalent antibodies (TvAb) wasevaluated in an ELISA assay with immobilized full-length VEGF165-Hisprotein (R&D Systems) and human ANG-2-His protein (R&D Systems) fordetection of bound bispecific antibody (FIG. 7). Only a bispecific<VEGF-ANG-2> TvAb is able to bind simultaneously to VEGF and ANG-2 andthus bridge the two antigens whereas monospecific “standard” IgG1antibodies should not be capable of simultaneously binding to VEGF andANG-2 (FIG. 7).

For this sake Falcon polystyrene clear enhanced microtiter plates werecoated with 100 μl 2 μg/mL recombinant human VEGF165 (R&D Systems) inPBS for 2 h at room temperature or over night at 4° C. The wells werewashed three times with 300 μl PBST (0.2% Tween 20) and blocked with 200μl 2% BSA 0.1% Tween 20 for 30 min at room temperature and subsequentlywashed three times with 300 μl PBST. 100 μL/well of a dilution series(40 pM-0.01 pM) of purified <VEGF-ANG-2> TvAb and as a reference <ANG-2>antibody Mab536 and VEGF> antibody G6-31 in PBS (Sigma) was added to thewells and incubated for 1 h on a microtiterplate shaker at roomtemperature. The wells were washed three times with 300 μl PBST (0.2%Tween 20) and bound antibody was detected by addition of 100 μl 0.5μg/ml human ANG-2-His (R&D Systems) in PBS. The wells were washed threetimes with 300 μl PBST (0.2% Tween 20) and bound ANG-2 was detected with100 μl 0.5 μg/mL<ANG-2>mIgG1-Biotin antibody (BAM0981, R&D Systems) for1 h at room temperature. Unbound detection antibody was washed away withthree times 300 μl PBST (0.2% Tween 20) and bound antibody was detectedby addition of 100 μl 1:2000 Streptavidin-POD conjugate (RocheDiagnostics GmbH, Cat. No. 11089153) 1:4 diluted in blocking buffer for1 h at room temperature. Unbound Streptavidin-POD conjugate was washedaway with three-six times 300 μl PBST (0.2% Tween 20) and boundStrepatavidin-POD conjugate was detected by addition of 100 μLABTS/well. Determination of absorbance was performed on a Tecan FluorSpectrometer at a measurement wavelength of 405 nm (reference wavelength492 nm).

Demonstration of Simultaneous Binding of Bispecific Tetravalent Antibody<VEGF-Ang-2> TvAb6 to VEGF-A and Ang-2 by Biacore

In order to further corroborate the data from the bridging ELISA anadditional assay was established to confirm simultaneous binding to VEGFand Ang-2 using surface plasmon resonance technology on a Biacore T100instrument according to the following protocol and analyzed using theT100 software package (T100 Control, Version 2.01, T100 Evaluation,Version 2.01, T100 Kinetics Summary, Version 1.01): Ang-2 was capturedwith a capture level of 2000-1700 RU in PBS, 0.005% (v/v) Tween20running buffer via a PentaHisAntibody (PentaHis-Ab BSA-free, Qiagen No.34660) that was immobilized on a CM5 chip via amine coupling (BSA-free).HBS-N buffer served as running buffer during coupling, activation wasdone by mixture of EDC/NHS. The PentaHis-Ab BSA-free Capture-Antibodywas diluted in coupling buffer NaAc, pH 4.5, c=30 μg/mL, finally stillactivated carboxyl groups were blocked by injection of 1 M Ethanolamine;ligand densities of 5000 and 17000 RU were tested. Ang-2 with aconcentration of 500 nM was captured by the PentaHis-Ab at a flow of 5μL/min diluted with running buffer+1 mg/mL BSA. Subsequently, <Ang-2,VEGF> bispecific antibody binding to Ang-2 and to VEGF was demonstratedby incubation with rhVEGF and formation of a sandwich complex. For thissake, bispecific <VEGF-Ang-2> TvAb6 was bound to Ang-2 at a flow of 50μL/min and a concentration of 100 nM, diluted with running buffer+1mg/mL BSA and simultaneous binding was detected by incubation with VEGF(rhVEGF, R&D-Systems Cat.-No, 293-VE) in PBS+0.005% (v/v) Tween20running buffer at a flow of 50 μL/min and a VEGF concentration of 150nM. Association time 120 sec, dissociation time 1200 sec. Regenerationwas done after each cycle at a flow of 50 μL/min with 2×10 mM Glycin pH2.0 and a contact time of 60 sec. Sensorgrams were corrected using theusual double referencing (control reference: binding of bispecificantibody and rhVEGF to capture molecule PentaHisAb). Blanks for each Abwere measured with rhVEGF concentration “0”. A scheme of the Biacoreassay is shown in FIG. 13. An alternative Biacore assay format is shownin FIG. 15.

Generation of HEK293-Tie2 Cell Line

In order to determine the interference of Angiopoietin-2 antibodies withANGPT2 stimulated Tie2 phosphorylation and binding of ANGPT2 to Tie2 oncells a recombinant HEK293-Tie cell line was generated. Briefly, apcDNA3 based plasmid (RB22-pcDNA3 Topo hTie2) coding for full-lengthhuman Tie2 (SEQ ID 108) under control of a CMV promoter and a Neomycinresistance marker was transfected using Fugene (Roche Applied Science)as transfection reagent into HEK293 cells (ATCC) and resistant cellswere selected in DMEM 10% FCS, 500 μg/ml G418. Individual clones wereisolated via a cloning cylinder, and subsequently analyzed for Tie2expression by FACS. Clone 22 was identified as clone with high andstable Tie2 expression even in the absence of G418 (HEK293-Tie2clone22). HEK293-Tie2 clone22 was subsequently used for cellular assays:ANGPT2 induced Tie2 phosphorylation and ANGPT2 cellular ligand bindingassay.

ANGPT2 Induced Tie2 Phosphorylation Assay

Inhibition of ANGPT2 induced Tie2 phosphorylation by ANGPT2 antibodieswas measured according to the following assay principle. HEK293-Tie2clone22 was stimulated with ANGPT2 for 5 minutes in the absence orpresence of ANGPT2 antibody and P-Tie2 was quantified by a sandwichELISA. Briefly, 2×105 HEK293-Tie2 clone 22 cells per well were grownover night on a Poly-D-Lysine coated 96 well-microtiter plate in 100 μlDMEM, 10% FCS, 500 μg/ml Geneticin. The next day a titration row ofANGPT2 antibodies was prepared in a microtiter plate (4-foldconcentrated, 75 μl final volume/well, duplicates) and mixed with 75 μlof an ANGPT2 (R&D systems #623-AN] dilution (3.2 μg/ml as 4-foldconcentrated solution). Antibodies and ANGPT2 were pre-incubated for 15min at room temperature. 100 μl of the mix were added to the HEK293-Tie2clone 22 cells (pre-incubated for 5 min with 1 mM NaV3O4, Sigma #S6508)and incubated for 5 min at 37° C. Subsequently, cells were washed with200 μl ice-cold PBS+1 mM NaV3O4 per well and lysed by addition of 120 μllysis buffer (20 mM Tris, pH 8.0, 137 mM NaCl, 1% NP-40, 10% glycerol, 2mM EDTA, 1 mM NaV3O4, 1 mM PMSF and 10 μg/ml Aprotinin) per well on ice.Cells were lysed for 30 min at 4° C. on a microtiter plate shaker and100 μl lysate were transferred directly into a p-Tie2 ELISA microtiterplate (R&D Systems, R&D #DY990) without previous centrifugation andwithout total protein determination. P-Tie2 amounts were quantifiedaccording to the manufacturer's instructions and IC50 values forinhibition were determined using XLfit4 analysis plug-in for Excel(Dose-response one site, model 205). IC50 values can be compared withinon experiment but might vary from experiment to experiment.

VEGF Induced HUVEC Proliferation Assay

VEGF induced HUVEC (Human Umbilical Vein Endothelial Cells, Promocell#C-12200) proliferation was chosen to measure the cellular function ofVEGF antibodies. Briefly, 5000 HUVEC cells (low passage number, <5passages) per 96 well were incubated in 100 μl starvation medium (EBM-2Endothelial basal medium 2, Promocell #C-22211, 0.5% FCS,Penicilline/Streptomycine) in a collagen I-coated BD Biocoat Collagen I96-well microtiter plate (BD #354407/35640 over night. Varyingconcentrations of antibody were mixed with rhVEGF (30 ngl/ml finalconcentration, BD #354107) and pre-incubated for 15 minutes at roomtemperature. Subsequently, the mix was added to the HUVEC cells and theywere incubated for 72 h at 37° C., 5% CO2. On the day of analysis theplate was equilibrated to room temperature for 30 min and cellviability/proliferation was determined using the CellTiter-Glo™Luminescent Cell Viability Assay kit according to the manual (Promega,#G7571/2/3). Luminescence was determined in a spectrophotometer.

Design of Tetravalent Bispecific and Tetravalent Monospecific Antibodies

The bispecific antibodies binding to VEGF (VEGF-A) and ANG-2(Angiopoietin-2) according to the invention comprise a firstantigen-binding site that binds to VEGF and a second antigen-bindingsite that binds to ANG-2. As first antigen-binding site binding to VEGF,e.g. the heavy chain variable domain of SEQ ID NO: 23, and the lightchain variable domains of SEQ ID NO: 24 which are both derived from thehuman phage display derived anti-VEGF antibody G6-31 which is describedin detail in Liang, W. C., et al., J Biol Chem. 281(2) (2006) 951-61 andin US 2007/0141065, can be used. Alternatively e.g. the secondantigen-binding site specifically binding to VEGF comprises the heavychain variable domains of SEQ ID NO: 7, or SEQ ID NO: 100, and the lightchain variable domains SEQ ID NO:8 or SEQ ID NO: 101 from the anti-VEGFantibodies <VEGF>bevacizumab and <VEGF>B20-4.1., preferably from<VEGF>bevacizumab.

As second antigen-binding site comprises the heavy chain variabledomains SEQ ID NO: 31, and the light chain variable domains SEQ ID NO:32 or SEQ ID NO: 32 with the mutations T92L, H93Q and W94T (Kabatnumbering), which are both derived from the human anti-ANG-2 antibody<ANG-2> Mab536 which is described in detail in Oliner, J., et al.,Cancer Cell. 6(5) (2004) 507-16, and in US 2006/0122370, can be used.Alternatively e.g. the second antigen-binding site specifically bindingto ANG-2 comprises the heavy chain variable domains of SEQ ID NO: 44,SEQ ID NO: 52, SEQ ID NO: 60, SEQ ID NO: 68, SEQ ID NO: 76, SEQ ID NO:84 or SEQ ID NO: 92, and the light chain variable domains SEQ ID NO: 45,SEQ ID NO: 53, SEQ ID NO: 61, SEQ ID NO: 69, SEQ ID NO: 77, SEQ ID NO:85, SEQ ID NO: 93 from the anti-ANG-2 antibodies <ANG-2> Ang2s_R3_LC03,<ANG-2>Ang2i_LC06, <ANG-2>Ang2i_LC07, <ANG-2> Ang2k_LC08, <ANG-2>Ang2s_LC09, <ANG-2> Ang2i_LC10, or <ANG-2> Ang2k_LC11, preferably from<ANG-2> Ang2i_LC06, or <ANG-2> Ang2k_LC08.

To generate agents that combine features of both antibodies, noveltetravalent bispecific antibody-derived protein entities wereconstructed. In these molecules, recombinant single-chain bindingmolecules of one antibody are connected via recombinant protein fusiontechnologies to the other antibody which was retained in the format of afull-length IgG1. This second antibody carries the desired secondbinding specificity.

By gene synthesis and recombinant molecular biology techniques, theheavy chain variable domain (VH) and the light chain variable domain(VL) of the respective antibody were linked by a glycine serine (G45)3or (G45)4 single-chain-linker to give a single chain Fv (scFv), whichwas attached to the C-terminus of the other antibody heavy chain using a(G)6- or (G45)3-linker.

In addition, cysteine residues were introduced in the VH (includingKabat position 44,) and VL (including Kabat position 100) domain of thescFv binding to ANG-2 or VEGF as described earlier (e.g. WO 94/029350;Reiter, Y., et al., Nature biotechnology (1996) 1239-1245; Young, N. M.,et al, FEBS Letters (1995) 135-139; or Rajagopal, V., et al., ProteinEngineering (1997) 1453-59).

All these molecules were recombinantly produced, purified andcharacterized and protein expression, stability and biological activitywas evaluated.

A summary of the bispecific antibody designs that were applied togenerate tetravalent bispecific <VEGF-ANG-2>, <ANG-2-VEGF> antibodiesand tetravalent monospecific <ANG-2> antibodies is given in Table 3. Forthis study, we use the term ‘TvAb’ to describe the various tetravalentprotein entities.

In order to obtain the bispecific tetravalent antibodies <VEGF-ANG-2>TvAb5 and TvAb6 the single chain Fv (scFv) binding to Angiopoietin-2derived from the heavy chain variable domain (VH) of SEQ ID NO: 31, andthe light chain variable domain (VL) of SEQ ID NO: 32 with the mutationsT92L, H93Q and W94T derived from the human anti-ANG-2 antibody <ANG-2>Mab536 was fused to the sequence corresponding to the C-terminus of theheavy chain vector of the human anti-VEGF antibody <VEGF> G6-31 of SEQID NO: 23 and co-expressed with the respective light chain expressionvector based on SEQ ID NO: 24. A representation of the designed formatsis shown in FIG. 1B and listed in Table 3.

In order to obtain the bispecific tetravalent antibodies TvAb9 andTvAb15 the single chain Fv (scFv) binding to VEGF derived from the heavychain variable domain (VH) of SEQ ID NO: 23, and the light chainvariable domain (VL) of SEQ ID NO: 24 derived from the human anti-VEGFantibody <VEGF> G6-31 were fused to the sequence corresponding to theC-terminus of the heavy chain vector of the human anti-ANG-2 antibody<ANG-2> Mab536 of SEQ ID NO: 31 and co-expressed with the respectivelight chain expression vector based on SEQ ID NO: 32. A representationof the designed formats is shown in FIG. 1B and listed in Table 3.

TABLE 3 The different bispecific tetravalent antibody formats with C-terminal scFv attachments and the corresponding TvAb-nomenclature. An“—” in the table means “not present” Position Molecule Name Variable ofscFv scFv (TvAb-nomen- scFv Domains attached Single- disulfide claturefor bispe- Antibody backbone derived VH and VL: to chain- Peptide-VH44/VL100 cific antibodies) derived from from SEQ ID NO: antibodylinker linker stabilized G6-31 <VEGF> — 23 + 24 — — — — (1000) G6-31Mab536 <ANG-2> — 31 + 32 — — — — (1000) Mab536 bevacizumab<VEGF>bevacizumab — 23 + 24 — — — — Ang2i_LC06 <ANG- — 52 + 53 — — — —(LC06) 2>Ang2i_LC06 Ang2k_LC06 <ANG- — 68 + 69 (LC08) 2>Ang2k_LC08 TvAb5<VEGF> <ANG-2> 23 + 24, C-term. (G4S)3 (G)6 — (2310) G6-31 Mab536 31 +32 HC with the mutations T92L, H93Q andW94T TvAb6 <VEGF> <ANG-2> 23 +24, C-term. (G4S)3 (G4S)3 scFv (2331) G6-31 Mab536 31 + 32 HC disulfidewith the VH44/ mutations VL100 T92L, stabilized H93Q andW94T TvAb9<ANG-2> <VEGF> 31 + 32, C-term. (G4S)3 (G4S)3 — (2330) Mab536 G6-31 and23 + HC 24 TvAb15 <ANG-2> <VEGF> 31 + 32, C-term. (G4S)4 (G4S)3 scFv(2431) Mab536 G6-31 and 23 + HC disulfide 24 VH44/ VL100 stabilizedTvAb-2441- bevacizumab LC06 7 + 8 and C-term. (G4S)4 (G4S)4 scFvbevacizumab- 52 + 53 HC disulfide LC06 VH44/ VL100 stabilized TvAb-2441-bevacizumab LC08 7 + 8 and C-term. (G4S)4 (G4S)4 scFv bevacizumab- 68 +69 HC disulfide LC08 VH44/ VL100 stabilized TvAb- bevacizumab LC06 7 + 8and N-term. (G4S)4 (G4S)2 scFv 3421_bevacizumab_LC06 52 + 53 HCdisulfide VH44/ VL100 stabilized TvAb- bevacizumab LC06 7 + 8 and C-term(G4S)4 (G4S)2 scFv 4421_bevacizumab_LC06 52 + 53 LC disulfide VH44/VL100 stabilized TvAb- bevacizumab LC06 7 + 8 and C-term (G4S)4 (G4S)6scFv 4461_bevacizumab_LC06 52 + 53 LC disulfide VH44/ VL100 stabilized

The TvAb formats are based e.g. on

-   -   a) aa) the human anti-VEGF antibody <VEGF> G6-31 and ab) two        single chain Fv (scFv) binding to Angiopoietin-2 derived from        the heavy chain variable domain (VH) of SEQ ID NO: 31, and the        light chain variable domain (VL) of SEQ ID NO: 32 with the        mutations T92L, H93Q and W94T, which are linked to the        C-terminus of the heavy chain of the anti-VEGF antibody <VEGF>        G6-31 (SEQ ID NO: 23); or    -   b) ba) the human anti-ANG-2 antibody <ANG-2> Mab536 and bb) two        single chain

Fv (scFv) binding to VEGF derived from the heavy chain variable domain(VH) of SEQ ID NO: 23, and the light chain variable domain (VL) of SEQID NO: 24, which are linked to the C-terminus of the heavy chain of theanti-ANG-2 antibody <ANG-2> Mab536 (SEQ ID NO: 31); or

-   -   c) ca) the human anti-VEGF antibody <VEGF> bevacizumab and cb)        two single chain Fv (scFv) binding to Angiopoietin-2 derived        from the heavy chain variable domain (VH) of SEQ ID NO: 52 or of        SEQ ID NO: 68, and the light chain variable domain (VL) of SEQ        ID NO: 53 or of SEQ ID NO: 69, which are linked to the        C-terminus of the heavy chain of the anti-VEGF antibody <VEGF>        bevacizumab (the Sequences of the resulting fusion peptide are        SEQ ID NO: 102 or SEQ ID NO: 103, which are co-expressed with        the light chain of bevacizumab SEQ ID NO: 104. (Alternatively        two single chain Fv (scFv) binding to Angiopoietin-2 can also be        linked to the C-terminus of the light chain or the N-terminus of        the heavy chain).    -   d) Alternatively to the single two single chain Fv (scFv) also        single chain Fab fragments can be used as described above (using        peptide connectors for the fusion to C or N-termini), in EP        Appl. No 09004909.9 and in Example 10.

Example 1 Expression & Purification of Bispecific Tetravalent Antibodies

<VEGF-ANG-2> TvAb5, TvAb6, TvAb-2441-bevacizumab-LC06 andTvAb-2441-bevacizumab-LC08

Light and heavy chains of the corresponding tetravalent bispecificantibodies TvAb5 and TvAb6 were constructed in genomic expressionvectors as described above. The plasmids were amplified in E. coli,purified, and subsequently transfected for transient expression ofrecombinant proteins in HEK293-F cells (utilizing Invitrogen's FreeStyle293 system). After 7 days, HEK 293-F cell supernatants were harvested,filtered and the bispecific antibodies were purified by protein A andsize exclusion chromatography. Homogeneity of all bispecific antibodyconstructs was confirmed by SDS-PAGE under non reducing and reducingconditions and analytical size exclusion chromatography. Under reducingconditions (FIG. 3), polypeptide heavy chains of <VEGF-ANG-2> TvAb6carrying the C-terminal scFv fusion showed upon SDS-PAGE apparentmolecular sizes of ca. 75 kDa analogous to the calculated molecularweights. Mass spectrometry confirmed the identity of the purifiedantibody constructs. Expression levels of all constructs were analysedby Protein A HPLC and were similar to expression yields of ‘standard’IgGs. Protein yields achieved up to 150 mg of TvAb6<VEGF-ANG-2> perliter of cell-culture supernatant as determined by Protein A HPLC.

Size exclusion chromatography analysis of the purified non-disulfidestabilized construct TvAb5 with C-terminal fused scFv at the heavy chainshowed that, compared to ‘standard’ IgGs, it had an increased tendencyto aggregate again after purification of monomeric antibody via sizeexclusion chromatography (so-called “daisy chain” phenomenon). Thisfinding has been supported by other examples (Rajagopal, V., et al.,Prot. Engin. (1997) 1453-1459; Kobayashi, H., et al, Nucl Med Biol.(1998) 387-393 or Schmidt, M., et al, Oncogene (1999) 18, 1711-1721)showing that molecules that contained scFvs that were not stabilized byinterchain disulfides between VH and VL exhibited an increased tendencyto aggregate and reduced yields. To address the problems withaggregation of such bispecific antibodies, disulfide-stabilization ofthe scFv moieties was applied. For that we introduced single cysteinereplacements within VH and VL of the scFv at defined positions(positions VH44/VL100 according to the Kabat numbering scheme). Thesemutations enable the formation of stable interchain disulfides betweenVH and VL, which in turn stabilize the resulting disulfide-stabilizedscFv module. Introduction of the VH44/VL100 disulfides in the scFv atthe C-terminus of the Fv in TvAb6 <VEGF-ANG-2> lead to a stabletetravalent antibody that showed no aggregation tendency any longerafter purification and remained in a monomeric state (FIG. 4A-C). Inaddition, TvAb6 <VEGF-ANG-2> showed no increase in aggregation tendencyupon repeated freeze-thaw cycles e.g. at the concentration applied forin vitro and in vivo of 3 mg/kg.

All other TvAb molecules described in Table 3 (e.g.TvAb-2441-bevacizumab-LC06 and TvAb-2441-bevacizumab-LC08) were preparedand analytically characterized analogously to the procedure described.

Example 2 Simultaneous Binding of Bispecific Tetravalent Antibody<VEGF-ANG-2> TvAb6, TvAb-2441-bevacizumab-LC06 andTvAb-2441-bevacizumab-LC08 to VEGF-A and ANG-2

The binding of the scFv modules and of the Fvs retained in theIgG-module of the different bispecific antibody formats were compared tothe binding of the ‘wildtype’ IgGs from which the binding modules andbispecific antibodies were derived. These analyses were carried out atequimolar concentrations by performing biochemical binding ELISAs and byapplying Surface Plasmon Resonance (Biacore).

For <VEGF-ANG-2> TvAb6 is was shown by VEGF binding ELISA as describedabove that it binds to VEGF comparable to its parent antibody G6-31 atan equimolar concentration of 0.625 pM (FIG. 5). This finding could beexpected as the Fv region of the TvAb is identical to that of G6-31. Theslight difference between <VEGF-ANG-2> TvAb6 and <VEGF> G6-31 is due tosmall differences in protein concentration and a slight stericinterference of the C-terminal scFv with binding of the <hFc>-PODdetection antibody and can be overcome by application of a <hk> POD(Biozol Cat. No. 206005) detection antibody like used for the ANG-2binding ELISA.

Using Biacore these findings were confirmed using a classicalconcentration series at 37° C. (FIG. 11). These data showed fastKon-rates k(a) of 4.7-4.8 E+6 1/(Ms), saturation was reached with thehighest concentrations of VEGF. Koff-rates reached limits of technicalspecification. (i.e. 5 x E-6 (s/s) probably due to still bivalentbinding (avidity effect) under this conditions as a consequence of thedimeric analyte rhVEGF, although a very low ligand density was usedresulting in final VEGF-response of 10-15 RU. Nevertheless, the kineticconstants of the different <VEGF> antibodies could be compared by thismethod and within the error of the method there was no significantdifference in the kinetic constants of the tetravalent bispecificantibody <VEGF-Ang-2> TvAb6 and the original antibody <VEGF> G6-31detectable. The kinetic constants for <VEGF-Ang-2> TvAb6 and <VEGF>G6-31 under these conditions were virtually identical by this method.Thus, it can be concluded that TvAb6 completely retained its VEGFbinding properties. Tab. 4 shows the respective kinetic constants andFIG. 12 shows the kinetic characteristics of the two <VEGF> antibodies<VEGF-Ang-2> TvAb6 and <VEGF> G6-31 in a K_(A)-K_(D) plot.

TABLE 14 Kinetic properties of <VEGF-Ang-2> TvAb6 and <VEGF> G6-31Measured at 37° C. K_(A) K_(D) t1/2 K_(D) Antibodies [1/(Ms)] [1/s][min] [M] <VEGF> G6-31 4.83E+06 9.33E−06 1237.8 1.93E−12 <VEGF-Ang-2>TvAb6 4.72E+06 7.24E−06 1596.7 1.53E−12

In a further experiment it was shown by ANG-2 binding ELISA using a<hk>-POD detection antibody (Biozol Catalogue No. 206005) as describedabove that <VEGF-ANG-2> TvAb6 binds to ANG-2 in a manner comparable tothat of Mab536 at an equimolar concentration of 0.039 pM (FIG. 6A). Thisshowed that the scFv module of TvAb6 retained its binding properties inthe TvAb construct.

In order to further corroborate this finding <ANG-2> Mab536 and<VEGF-ANG-2> TvAb6 were immobilized by a secondary antibody on a BiacoreCM5 chip and binding kinetics to human ANG-2 were determined. Due toheterogeneity of the ANG-2 preparation no 1:1 binding can be observed;K_(D) values are thus only relative estimations. The Biacore analysisshowed that <VEGF-ANG-2> TvAb6 has an estimated K_(D) value of 4.4 nMfor ANG-2. In comparison, Mab536 has an estimated K_(D) value of 1.6 nM.Within the error of the method no difference in binding mode andaffinities between <ANG-2> Mab536 and <VEGF-ANG-2> TvAb6 could beobserved (FIG. 6B). Thus, it can be concluded that the scFv module ofTvAb6 completely retained its binding properties in the TvAb construct.

In order to prove that <VEGF-ANG-2> TvAb6 was able to bindsimultaneously to VEGF and ANG-2 bridging ELISA assays and Biacoreassays as described above were applied.

By applying the VEGF-ANG-2-bridging ELISA described above it was shownthat only <VEGF-ANG-2> TvAb6 was able to bind simultaneously to VEGF andANG-2 at an equimolar concentration of 0.625 pM whereas the monospecific“standard” IgG1 antibodies <ANG-2> Mab536 and <VEGF> G6-31 were notcapable of simultaneously binding to VEGF and ANG-2 (FIG. 7).

FIG. 14 shows the respective data from the Biacore assay. Simultaneousbinding of both antigens Ang-2 and VEGF could be shown for thetetravalent bispecific antibody <VEGF-Ang-2> TvAb6. Negative controlswere as expected: The monospecific antibody <Ang-2> Mab536 showed onlybinding to Ang-2, but no VEGF-binding. The monospecific antibody <VEGF>G6-31 showed binding to VEGF but no binding to Ang-2 at all (data notshown). From the relative response units of the tetravalent bispecificantibody <VEGF-Ang-2> TvAb6 binding to the Ang-2 coated surface, andsubsequent binding to dimeric VEGF binding the stochiometry could becalculated to be in the range from 1:1 to 1:1.4. Taken together, byapplying the described ELISA and Biacore assays it was shown that only<VEGF-Ang-2> TvAb6 was able to bind simultaneously to VEGF and Ang-2whereas the monospecific “standard” IgG1 antibodies <Ang-2> Mab536 and<VEGFY G6-31 were not capable of simultaneously binding to VEGF andAng-2 (FIG. 15).

Similar results were obtained with the constructsTvAb-2441-bevacizumab-LC06 and TvAb-2441-bevacizumab-LC08 in ananalogous Biacore assay shown in FIG. 15A. Binding of the antibodies tothe antigen e.g. human ANG-2 and VEGF were investigated by surfaceplasmon resonance using a BIACORE T100 instrument (GE HealthcareBiosciences AB, Uppsala, Sweden). Briefly, for affinity measurementsgoat<hIgG-Fc□> polyclonal antibodies were immobilized on a CM4 chip viaamine coupling for presentation of the bispecific antibodies againsthuman ANG-2 and VEGF. Binding was measured in HBS buffer (HBS-P (10 mMHEPES, 150 mM NaCl, 0.005% Tween 20, ph 7.4), 25° C. Purified ANG-2-His(R&D systems or in house purified) was added in various concentrationsbetween 6.25 nM and 200 nM in solution. Association was measured by anANG-2-injection of 3 minutes; dissociation was measured by washing thechip surface with FIBS buffer for 3 minutes and a K_(D) value wasestimated using a 1:1 Langmuir binding model. Due to heterogenity of theANG-2 preparation no 1:1 binding could be observed; K_(D) values arethus only relative estimations.

VEGF (R&D systems) was added in various concentrations between 6.25 nMand 200 nM in solution. Association was measured by an VEGF-injection of3 minutes; dissociation was measured by washing the chip surface withHBS buffer for 3 minutes and a K_(D) value was estimated using a 1:1Langmuir binding model.

The order of injection of the binding partners can switched, first VEGFand then Ang2 or vice versa.

Negative control data (e.g. buffer curves) were subtracted from samplecurves for correction of system intrinsic baseline drift and for noisesignal reduction. Biacore T100 Evaluation Software version 1.1.1 wasused for analysis of sensorgrams and for calculation of affinity data.

Antibody Affinity hAng-2 Affinity hVEGF TvAb-2441-bevacizumab-LC06 2.3nM 0.35 nM TvAb-2441-bevacizumab-LC08 0.7 nM 0.34 nM G6-31 — <0.1 nMMAb536 3 nM — bevacizumab — 0.59 nM

Finally, simultaneous binding of TvAb-2441-bevacizumab-LC06 andTvAb-2441-bevacizumab-LC08 could be shown by incubating with ANGPT2 andVEGF in a consecutive manner. As shown in FIG. 15B ANGPT2 and VEGF canbind simultaneously to the bispecific antibodies.

Example 3 In Vivo Efficacy of Disulfide-Stabilized BispecificTetravalent Antibody <VEGF-ANG-2> TvAb6 in Comparison to <ANG-2> Mab536,<VEGF> G6-31 and the Combination of Mab536 and G6-31 in the StagedSubcutaneous Colo205 Xenograft Model in Scid Beige Mice

The purified disulfide-stabilized <VEGF-ANG-2> TvAb6 (n00.2331 see Table3) was compared to the antibodies <ANG-2> Mab536, <VEGF> G6-31 and thecombination of <ANG-2> Mab536 and <VEGF> G6-31 in two stagedsubcutaneous Colo205 xenograft model studies (Ang2_PZ_Colo205_003 andAng2_PZ_Colo205_005) in female Scid beige mice at different doses.

Antibodies: <ANG-2> Mab536 was provided as frozen stock solution (c=4.5mg/mL), <VEGF> G6-31 was provided as frozen solution (c=0.6 mg/mL) and<VEGF-ANG-2> TvAb6 was provided as frozen stock solution (c=0.5 mg/mL)in 20 mM Histidine, 140 mM NaCl, pH 6.0. Antibody solution was dilutedappropriately in PBS from stock prior injections where required and PBSwas applied as vehicle.

Cell lines and culture conditions: Colo205 human colorectal cancer cellswere originally obtained from ATCC and after expansion deposited in theRoche Penzberg internal cell bank. Tumor cell line was routinelycultured in RPMI 1640 medium (PAA, Laboratories, Austria) supplementedwith 10% fetal bovine serum (PAA Laboratories, Austria) and 2 mML-glutamine, at 37° C. in a water-saturated atmosphere at 5% CO₂.Passage 2-5 was used for transplantation.

Animals: Female SCID beige mice; age 4-5 weeks at arrival (purchasedfrom Charles River Germany) were maintained under specific-pathogen-freecondition with daily cycles of 12 h light/12 h darkness according tocommitted guidelines (GV-Solas; Felasa; TierschG). Experimental studyprotocol was reviewed and approved by local government. After arrivalanimals were maintained in the quarantine part of the animal facilityfor one week to get accustomed to the new environment and forobservation. Continuous health monitoring was carried out on regularbasis. Diet food (Provimi Kliba 3337) and water (acidified pH 2.5-3)were provided ad libitum. Age of mice at start of the study was about 10weeks.

Monitoring: Animals were controlled daily for clinical symptoms anddetection of adverse effects. For monitoring throughout the experimentbody weight of animals was documented and tumor volume was measured bycaliper after staging.

Tumor cell injection: At day of injection Colo205 cells werecentrifuged, washed once and resuspended in PBS. After an additionalwashing with PBS cell concentration and cell size were determined usinga cell counter and analyzer system (Vi-CELL, Beckman Coulter). Forinjection of Colo205 cells, the final titer was adjusted to 5.0×10E7cells/ml, viability ca. 90%. Subsequently 100 μl of this suspensioncorresponding to 2.5*106 cells per animal was injected s.c. into theright flank of the mice.

Treatment of animals started at day of randomisation, 16 days after celltransplantation (study Ang2_PZ_Colo205_003) and 14 days after celltransplantation (study Ang2_PZ_Colo205_005) at a mean tumor volume of100 mm3 or 150 mm3, respectively.

Dose Schedule Until Day 74 (See FIG. 8A) of Study Ang2_PZ_Colo205_003:

Route/ Cumu- No of Mode of No of lative ani- Com- Dose adminis- treat-Dose Group mals pound (mg/kg) tration ments mg/kg 1 10 Vehicle i.p. once4 weekly 2 10 <VEGF> 6 mg/kg i.p. once 8 48 G6-31 weekly 3 10 <ANG-2> 6mg/kg i.p. once 8 48 Mab536 weekly 4 10 <VEGF> 5 mg/kg + i.p. once 8 40G6-31 + 6 mg/kg weekly <ANG-2> i.p. once 8 48 Mab536 weekly 5 10<VEGF-ANG- 7 mg/kg i.p. once 8 56 2> weekly TvAb6

In the study Ang2_PZ_Colo205_003<VEGF-ANG-2> TvAb6 was by mistakeunderdosed with respect to an equimolar ratio. The dose of <VEGF-ANG-2>TvAb6 was adjusted in study Ang2_PZ_Colo205_005 so that the animalsreceived an equimolar ratio of ANG-2 and VEGF binding sites by<VEGF-ANG-2> TvAb6 as well as the combination of <VEGF> G6-31 and<ANG-2> Mab536.

Tumor growth inhibition until Day 74 (see FIG. 8a ) Ang2_PZ_Colo205_003study: <VEGF-ANG-2> TvAb6 at a dose of 7 mg/kg exhibited efficacycomparable to that of the combination of <VEGF> G6-31 at 5 mg/kg and<ANG-2> Mab536 at 6 mg/kg and <VEGF> G6-31 as single agent at a dose of6 mg/kg (FIG. 8A) and was superior to single agent <ANG-2> Mab536 at adose of 6 mg/kg. As the subcutaneous Colo205 model is very responsive tothe <VEGF> G6-31 antibody that blocks human as well as murine VEGFresulting in almost complete tumor growth inhibition <VEGF-ANG-2> TvAb6could thus not be differentiated from G6-31 as single agent (6 mg/kg)under the chosen experimental conditions, while <VEGF-ANG-2> TvAb6showed a comparable inhibition like the combination of <ANG-2> Mab536and <VEGF> G6-31 at a clearly lower cumulative dose (<VEGF-ANG-2>TvAb6=56 mg/kg antibody compared to the combination of <ANG-2> Mab536and <VEGF> G6−31=40+48=88 mg/kg antibody).

Dose schedule of Study until Day 63 Ang2_PZ_Colo205_005:

Route/ No of Mode of No of Cumu- ani- Com- Dose adminis- treat- lativeGroup mals pound (mg/kg) tration ment Dose 6 10 Vehicle i.p. once 6weekly 7 10 <VEGF> 3 i.p. once 7 21 mg/kg G6-31 weekly 8 10 <VEGF> 3i.p. once 7 21 mg/kg G6-31 + 3 weekly 7 21 mg/kg <ANG-2> i.p. onceMab536 weeklyl 9 10 <ANG-2> 3 i.p. once 7 21 mg/kg Mab536 weekly 10 10<VEGF- 4 i.p. once 7 28 mg/kg ANG-2> weekly TvAb6

Tumor Growth Inhibition Until Day 63 Ang2_PZ_Colo205_005 Study:

<VEGF-ANG-2> TvAb6 at a dose of 4 mg/kg exhibited efficacy comparable tothat of the combination of <VEGF> G6-31 and <ANG-2> Mab536 at 3 mg/kgeach and was superior to either single agent <VEGF> G6-31 as well as<ANG-2> Mab536 at a dose of 3 mg/kg (FIG. 8B). This is the first exampleshowing that at a lower dose (with respect to the summarizedconcentration of antibody—the cumulative dose of the combination is21+21=42 m/kg versus 28 mg/kg of the bispecific antibody TvAb6) abispecific antibody targeting VEGF and ANG-2 can result in stronganti-tumor efficacy comparable to the combination of the respectivesingle agents blocking VEGF and ANG-2 and superior to either singleagent.

Example 4 Blocking of VEGF-Induced Tube Formation

In order to confirm that the anti-VEGF related activities were retainedin the bispecific tetravalent <VEGF-ANG-2> TvAb6 is was shown in a VEGFinduced tube formation assay AngioKit TCS CellWorks (Cell Systems) that<VEGF-ANG-2> TvAb6 mediated dose dependent inhibition of tube formationwas comparable to inhibition of tube formuation when the monospecificantibody <VEGF> G6-31 was used. The AngioKit TCS CellWorks assay wasperformed according to the following procedure: Cells were stimulatedeach time with 2 ng/ml VEGF before treatment with antibodies on day 1,4, 7 and 9. Vascular tubes were visualized by staining of endothelialcells using a CD31-PE antibody (BD Pharmingen #555446) on day 11.Pictures were taken at a magnification of 4× and values for tube lengthand number of branch points were quantitatively analysed using theAngiogenesis Tube Formation Application Module in MetaMorph (MolecularDevices). Values and standard deviation were calculated by duplicatesand analysis of 4 pictures per specimen. FIG. 9 shows the respectiveresults and FIGS. 10 A and B the quantitative analysis. Angiopietin-2has no influence on tube formation and thus inhibition of ANG-2 was notstudied in this assay. The data show that the bispecific <VEGF-ANG-2>TvAb6 and the monospecific <VEGF> G6-31 antibodies are equallyefficacious in inhibiting VEGF stimulated tube formation.

Example 5 Tie2 Phosphorylation

In order to confirm that the anti-ANGPT2 related activities wereretained in the bispecific tetravalent <VEGF-ANGPT2> antibodiesTvAb-2441-bevacizumab-LC06 and TvAb-2441-bevacizumab-LC08, it was shownthat TvAb-2441-bevacizumab-LC06 and TvAb-2441-bevacizumab-LC08 interferewith ANGPT2 stimulated Tie2 phosphorylation in a comparable manner astheir mother clones LC06 and LC08 in the ANGPT2 stimulated Tie2phosphorylation assay as described above.

In a first experiment both bispecific antibodiesTvAb-2441-bevacizumab-LC06 and TvAb-2441-bevacizumab-LC08 showed adose-dependent interference with ANGPT2 stimulated Tie2 phosphorylationwith IC50 values comparable to those of the mother clones LC06 and LC08as shown in FIG. 16A. TvAb-2441-bevacizumab-LC06 interfered with ANGPT2stimulated Tie2 phosphorylation with a IC50 value of approx. 721 ng/ml,whereas LC06 interfered with ANGPT2 stimulated Tie2 phosphorylation witha IC50 value of approx. 508 ng/ml. TvAb-2441-bevacizumab-LC08 interferedwith ANGPT2 stimulated Tie2 phosphorylation with a IC50 value of approx.364 ng/ml, whereas LC08 interfered with ANGPT2 stimulated Tie2phosphorylation with a IC50 value of approx. 499 ng/ml.

In a second experiment both bispecific antibodiesTvAb-2441-bevacizumab-LC06 and TvAb-2441-bevacizumab-LC08 showed adose-dependent interference with ANGPT2 stimulated Tie2 phosphorylationwith IC50 values comparable to those of the mother clones LC06 and LC08as shown in FIG. 16B. TvAb-2441-bevacizumab-LC06 interfered with ANGPT2stimulated Tie2 phosphorylation with a IC50 value of approx. 488 ng/ml,whereas LC06 interfered with ANGPT2 stimulated Tie2 phosphorylation witha IC50 value of approx. 424 ng/ml. TvAb-2441-bevacizumab-LC08 interferedwith ANGPT2 stimulated Tie2 phosphorylation with a IC50 value of approx.490 ng/ml, whereas LC08 interfered with ANGPT2 stimulated Tie2phosphorylation with a IC50 value of approx. 399 ng/ml.

Taken together these data show that the bispecific tetravalent<VEGF-ANGPT2> antibodies TvAb-2441-bevacizumab-LC06 andTvAb-2441-bevacizumab-LC08 interfere with ANGPT2 stimulated Tie2phosphorylation in a manner comparable to their mother clones LC06 andLC08 within the error of this cellular assay.

Example 6 Inhibition of huANG-2 Binding to Tie-2 (ELISA)

The interaction ELISA was performed on 384 well microtiter plates(MicroCoat, DE, Cat. No. 464718) at RT. After each incubation stepplates were washed 3 times with PBST. ELISA plates were coated with 0.5μg/ml Tie-2 protein (R&D Systems, UK, Cat. No. 313-TI) for at least 2hours (h). Thereafter the wells were blocked with PBS supplemented with0.2% Tween-20 and 2% BSA (Roche Diagnostics GmbH, DE) for 1 h. Dilutionsof purified antibodies in PBS were incubated together with 0.2 μg/mlhuAngiopoietin-2 (R&D Systems, UK, Cat. No. 623-AN) for 1 h at RT. Afterwashing a mixture of 0.5 μg/ml biotinylated anti-Angiopoietin-2 cloneBAM0981 (R&D Systems, UK) and 1:3000 diluted streptavidin HRP (RocheDiagnostics GmbH, DE, Cat. No. 11089153001) was added for 1 h.Thereafter the plates were washed 6 times with PBST. Plates weredeveloped with freshly prepared ABTS reagent (Roche Diagnostics GmbH,DE, buffer #204 530 001, tablets #11 112 422 001) for 30 minutes at RT.Absorbance was measured at 405 nm.

Summary Data for Ang2 Interaction ELISA

AVG IC50 <VEGF-ANG-2> bispecific antibody (ng/ml) (or monospecificparent antibodies) hANG2 STDEV <VEGF-ANG-2> G6_31 >20000TvAb-2441_G6_31_Ang2i_LC06 75 39 TvAb-2441_G6_31_Ang2k_LC08 66 31TvAb-2441_bevacizumab_LC06 44 8 TvAb-2441_bevacizumab_LC08 42 11<ANG-2>Mab 536 15 8 <VEGF>Bevacizumab >20000 TvAb-3421_bevacizumab_LC0631 1 TvAb-4421_bevacizumab_LC06 35 17 TvAb-4461_bevacizumab_LC06 46 10

Example 7 Inhibition of hVEGF Binding to hVEGF Receptor (ELISA)

The test was performed on 384 well microtiter plates (MicroCoat, DE,Cat. No. 464718) at RT. After each incubation step plates were washed 3times with PBST. At the beginning, plates were coated with 0.5 μg/mlhVEGF-R protein (R&D Systems, UK, Cat. No. 321-FL) for at least 2 hours(h). Thereafter the wells were blocked with PBS supplemented with 0.2%Tween-20 and 2% BSA (Roche Diagnostics GmbH, DE) for 1 h. Dilutions ofpurified antibodies in PBS were incubated together with 0.15 μg/mlhuVEGF121 (R&D Systems, UK, Cat. No. 298-VS) for 1 h at RT. Afterwashing a mixture of 0.5 μg/ml anti VEGF clone Mab923 (R&D Systems, UK)and 1:2000 horse radish peroxidase (HRP)-conjugated F(ab′)2 anti mouseIgG (GE Healthcare, UK, Cat. No. NA9310V) was added for 1 h. Thereafterthe plates were washed 6 times with PBST. Plates were developed withfreshly prepared ABTS reagent (Roche Diagnostics GmbH, DE, buffer #204530 001, tablets #11 112 422 001) for 30 minutes at RT. Absorbance wasmeasured at 405 nm.

Summary Data for VEGF Interaction ELISA

AVG IC50 (ng/ml) <VEGF-ANG-2> bispecific antibody VEGF STDEV<VEGF-ANG-2> G6_31 1431 130 TvAb-2441_G6_31_Ang2i_LC06 1654 213TvAb-2441_G6_31_Ang2k_LC08 1392 184 TvAb-2441_bevacizumab_LC06 2831 503TvAb-2441_bevacizumab_LC08 2305 972 TvAb-<ANG-2>Mab 536 >20000TvAb-<VEGF>Bevacizumab 1584 357 TvAb-3421_bevacizumab_LC06 2660 284TvAb-4421_bevacizumab_LC06 1980 1319 TvAb-4461_bevacizumab_LC06 1677 394

Example 8 HUVEC Proliferation

In order to confirm that the anti-VEGF related activities were retainedin the bispecific tetravalent <VEGF-ANG2>antibodiesTvAb-2441-bevacizumab-LC06 and TvAb-2441-bevacizumab-LC08 it was shownthat TvAb-2441-bevacizumab-LC06 and TvAb-2441-bevacizumab-LC08 interferewith VEGF-induced HUVEC proliferation in a comparable manner as theirmother clones LC06 and LC08 in the VEGF-induced HUVEC proliferationassay as described above.

FIG. 18 shows that indeed TvAb-2441-bevacizumab-LC06 andTvAb-2441-bevacizumab-LC08 interfere in a concentration dependent mannerwith VEGF-induced HUVEC proliferation comparable to the parentalantibody bevacizumab.

Example 9

ELISA Binding assay to human ANG-1 and to human ANG-2

The binding of parent <ANG-2> antibodies Ang2i-LC06, Ang2i-LC07 andAng2k-LC08 to human ANG-1 and human ANG-2 was determined in an ANG-1 orANG-2 binding ELISA as described above (see Comparative binding to ANG-1and ANG-2 (ANG-1 and ANG-2 binding ELISA)). Briefly, the ELISA-typeassay is based on the immobilization of human wild-type Angiopoieti-1 or-2 in a microtiter plate. Binding of an antibody directed against theimmobilized ANG-1 or ANG-2 is measured via an <human Fc> (anti-IgG)antibody with a POD conjugate. A dilution series of the <ANG-2> antibodyallows to determine an EC₅₀ concentration. As a reference the humananti-ANG-2 antibody <ANG-2> antibody Mab536 (Oliner et al., Cancer Cell.2004 November; 6(5):507-16, US 2006/0122370) was used.

The determined EC50 concentrations are summarized in the table below.

hANG-1 binding hANG-2 binding Antibody EC50 EC50 <ANG-2>MAb536 2538ng/mL 133 ng/mL <ANG-2>Ang2i-LC06 >8000 ng/mL 84 ng/mL<ANG-2>Ang2i-LC07 >8000 ng/mL 3006 ng/mL <ANG-2>Ang2i-LC08 4044 ng/mL105 ng/mL

All antibodies specifically bind to ANG-2. MAb536 and Ang2k-LC08 showalso specific binding towards ANG-1, whereas Ang2i-LC06 and Ang2i-LC07do not specifically bind to ANG-1 as they have an EC50-value of above8000 ng/ml (detection limit).

Example 10 Expression & Purification of Bispecific, Tetravalent SingleChain Fab <VEGF-ANG-2> Antibody Molecules scFAb-Bevacizumab-LC06-2620,scFab-Bevacizumab-Ang2i-LC06-2640 and scFab-Bevacizumab-Ang2i-LC06-2641

Analogous to the procedures described in Example 1 and in the materialsand methods above, the bispecific, tetravalent single chain Fab<VEGF-ANG-2> antibody molecules scFAb-bevacizumab-LC06-2620,scFab-bevacizumab-LC06-2640 and scFab-bevacizumab-LC06-2641, all threebased on <VEGF> bevacizumab and <ANG-2> Ang2i-LC06, were expressed andpurified. Binding affinities and other properties were determined asdescribed in the Examples above. The relevant (eventually modified)light and heavy chains amino acid sequences of these bispecificantibodies are given in SEQ ID NO: 109-110(scFAb-bevacizumab-LC06-2620), in SEQ ID NO: 111-112(scFAb-bevacizumab-LC06-2640) and in SEQ ID NO: 113-114(scFAb-bevacizumab-LC06-2641).

scFAb- scFAb- scFAb- Bevacizumab- Bevacizumab- Bevacizumab- Ang2i-LC06-LC06-Ang2i- Key data LC06-2620 2640 2641 Expression (Yield) 29 μg/mL 27μg/mL 18 μg/mL Purification (Yield, 21 mg, 57% 19 mg, 86% 12 mg, 90%Prot. A. homog.) Homogeneity after 98% 98% 99% preparative SEC FunctionhANG-2 affinity 1.9E−9M 1.8E−9M 1.9E−9M (Biacore) hVEGF affinity  1E−10M   1E−10M   1E−10M (Biacore)

Example 11 Expression & Purification of Bispecific, Trivalent SingleChain Fab <VEGF-ANG-2> Antibody Molecule Bevacizumab-LC06-KiH-C-scFab

Analogous to the procedures described in Example 1 and in the materialsand methods above, the bispecific, trivalent single chain Fab<VEGF-ANG-2> antibody molecule bevacizumab-LC06-KiH-C-scFab based on<VEGF> bevacizumab and <ANG-2> Ang2i-LC06 were expressed and purified.Binding affinities and other properties were determined as described inthe Examples above. The relevant (eventually modified) light and heavychains amino acid sequences of this bispecific antibody are given in SEQID NO: 115-117 (bevacizumab-LC06-KiH-C-scFab).

Bevacizumab- LC06-KiH-C- Key data scFab Expression (Yield) 15 μg/mLPurification (Yield, 4.8 mg, 91% Prot. A. homog.) Homogeneity after 97%preparative SEC Function hANG-2 affinity 4.4E−9M (Biacore) hVEGFaffinity   1E−10M (Biacore)

Example 12 Expression & Purification of Bispecific, Trivalent<VEGF-ANG-2> Antibody Molecule Bevacizumab-LC06-C-Fab-6CSS

Analogous to the procedures described in Example 1 and in the materialsand methods above (see also, the bispecific, trivalent <VEGF-ANG-2>antibody molecule bevacizumab-LC06-C-Fab-6CSS based on <VEGF>bevacizumab and <ANG-2> Ang2i-LC06 were expressed and purified. Bindingaffinities and other properties were determined as described in theExamples above. Bispecific, trivalent antibody molecules of this formatin general are described in EP Appl. No 09005108.7. The relevant(eventually modified) light and heavy chains amino acid sequences ofthis bispecific <VEGF-ANG-2> antibody are given in SEQ ID NO: 118-120(bevacizumab-LC06-C-Fab-6CSS).

scFAb- scFAb- scFAb- Bevacizumab- Bevacizumab- Bevacizumab- Key dataLC06-2620 LC06-2640 LC06-2641 Expression (Yield) 29 μg/mL 27 μg/mL 18μg/mL Purification (Yield, 21 mg, 57% 19 mg, 86% 12 mg, 90% Prot. A.homog.) Homogeneity after 98% 98% 99% preparative SEC Function hANG-2affinity 1.9E−9M 1.8E−9M 1.9E−9M (Biacore) hVEGF affinity   1E−10M  1E−10M   1E−10M (Biacore)

Example 13 Expression & Purification of Bispecific, Bivalent DomainExchanged <VEGF-ANG-2> Antibody Molecules Bevacizumab-LC06-CH1-CL,Bevacizumab-LC06-VH-VL and Bevacizumab-LC06-VH-VL-SS

Analogous to the procedures described in Example 1 and in the materialsand methods above, the bispecific, bivalent domain exchanged<VEGF-ANG-2> antibody molecules bevacizumab-LC06-CH1-CL (CH-CL exchangeas described in WO 2009/080253), bevacizumab-LC06-VH-VL (VH-VL exchangeas described in WO 2009/080252) and bevacizumab-LC06-VH-VL-SS (VH-VLexchange as described in WO 2009/080252 and an additional introducedVH44 VL100 disulfide bridge) based on <VEGF> bevacizumab and <ANG-2>Ang2i-LC06 were expressed and purified. Binding affinities and otherproperties were determined as described in the Examples above. Therelevant (eventually modified) light and heavy chains amino acidsequences of these bispecific antibodies are given in SEQ ID NO: 121-124(bevacizumab-LC06-CH1-CL), in SEQ ID NO: 125-128(Bevacizumab-LC06-VH-VL) and in SEQ ID NO: 129-132(bevacizumab-LC06-VH-VL-SS).

Bevacizumab- Bevacizumab- Bevacizumab- LC06-CM- LC06-CM- LC06-VH-VL- Keydata CH1-CL VH-VL SS Expression (Yield) 87 μg/mL 44 μg/mL 65 μg/mLPurification (Yield, 50 mg, 62% 22 mg, 95% 91 mg, 74% Prot. A. homog.)Homogeneity after 84% >99% 95% preparative SEC Function hANG-2 affinity1.3E−9M 2.1E−9M 1.46E−9M (Biacore) hVEGF affinity   1E−10M   1E−10M   1E−10M (Biacore)

Example 14 Expression & Purification of Bispecific, Bivalent ScFab-FcFusion <VEGF-ANG-2> Antibody Molecules Bevacizumab-LC06-N-scFab andBevacizumab-LC06-N-scFabSS

Analogous to the procedures described in Example 1 and in the materialsand methods above, the bispecific, bivalent ScFab-Fc fusion <VEGF-ANG-2>antibody molecules bevacizumab-LC06-N-scFab andbevacizumab-LC06-N-scFabSS based on <VEGF> bevacizumab and <ANG-2>Ang2i-LC06 were expressed and purified. Binding affinities and otherproperties were determined as described in the Examples above. Therelevant modified heavy chains amino acid sequences of these bispecificantibodies are given in SEQ ID NO: 133-134 (bevacizumab-LC06-N-scFab),and in SEQ ID NO: 135-136 (bevacizumab-LC06-N-scFabSS).

Bevacizumab- LC06-N- Bevacizumab- Key data scFab LC06-N-scFabSSExpression (Yield) 62 μg/mL Purification (Yield, 43% Prot. A. homog.)Function hANG-2 affinity 1 nM (Biacore) hVEGF affinity 1 nM (Biacore)

Example 15 Inhibition of hVEGF Binding to hVEGF Receptor (ELISA),Blocking of VEGF-Induced Tube Formation, Inhibition of huANG-2 Bindingto Tie-2 (ELISA), Tie2 Phosphorylation, and HUVEC Proliferation of theBispecific, <VEGF-ANG-2> Antibody Molecules of Examples 10 to 14

Inhibition of hVEGF binding to hVEGF Receptor (ELISA), blocking ofVEGF-induced tube formation, Inhibition of huANG-2 binding to Tie-2(ELISA), Tie2 phosphorylation, and HUVEC proliferation of thebispecific, <VEGF-ANG-2> antibody molecules of Examples 10 to 14 can bedetermined analogously to the procedures described in Materials andMethods and Examples 4 to 9 above.

Example 16 In Vivo Efficacy of Bispecific Antibody <VEGF-ANG-2> Antibodyin Comparison to <ANG-2> ANG2i-LC06, and the Combination of <ANG-2>ANG2i-LC06 and Bevacizumab in the Refractory Colo205 Xenograft Model inScid Beige Mice (after Resistance to Bevacizumab Treatment) Cell Linesand Culture Conditions:

Colo205 human colorectal cancer cells were originally obtained from ATCCand after expansion deposited in the Roche Penzberg internal cell bank.Tumor cell line was routinely cultured in RPMI 1640 medium (PAA,Laboratories, Austria) supplemented with 10% fetal bovine serum (PAALaboratories, Austria) and 2 mM L-glutamine, at 37° C. in awater-saturated atmosphere at 5% CO₂. Passage 2-5 was used fortransplantation.

Animals:

Female SCID beige mice; age 4-5 weeks at arrival (purchased from CharlesRiver Germanyd), were maintained under specific-pathogen-free conditionwith daily cycles of 12 h light/12 h darkness according to committedguidelines (GV-Solas; Felasa; TierschG). Experimental study protocol wasreviewed and approved by local government. After arrival animals weremaintained in the quarantine part of the animal facility for one week toget accustomed to new environment and for observation. Continuous healthmonitoring was carried out on regular basis. Diet food (Provimi Kliba3337) and water (acidified pH 2.5-3) were provided ad libitum. Age ofmice at start of the study was about 10 weeks.

Tumor Cell Injection:

At the day of injection, tumor cells were harvested (trypsin-EDTA) fromculture flasks (Greiner) and transferred into 50 ml culture medium,washed once and resuspended in PBS. After an additional washing stepwith PBS and filtration (cell strainer; Falcon 0100 μm) the final celltiter was adjusted to 2.5×10⁷/ml. Tumor cell suspension was carefullymixed with transfer pipette to avoid cell aggregation. After this, cellsuspension was filled into a 1.0 ml tuberculin syringe (Braun Melsungen)using a wide needle (1.10×40 mm); for injection needle size was changed(0.45×25 mm) and for every injection a new needle was used. Anesthesiawas performed using a Stephens inhalation unit for small animals withpreincubation chamber (plexiglas), individual mouse nose-mask (silicon)and not flammable or explosive anesthesia compound Isoflurane(cp-pharma) in a closed circulation system. Two days before injectioncoat of the animals were shaved and for cell injection skin ofanaesthetized animals was carefully lifted up with an anatomic forcepsand 100 μl cell suspension (=2.5×10⁶ cells) was injected subcutaneouslyin the right flank of the animals.

Treatment of Animals Pretreatment:

Animal treatment started 14 days after cell transplantation (studyAng2_PZ_Colo205_008) at a mean tumor volume of 100 mm³ to 150 mm³,respectively. Mice were treated once weekly with bevacizumab (10 mg/kg)for a time period of 5 weeks.

Secondary Treatment:

Thereafter mice were randomized for 2^(nd) treatment and divided to fourgroups with 10 mice in each group. Tumor volume at start of secondarytreatment at day 51 was in the range from 336 to 341 mm³. Mice weretreated once weekly i.p. with the different compounds as indicated infollowing table.

Route/ Cumu- No of Dose Mode of No of lative ani- Com- (mg/kg) adminis-treat- dose Group mals pound (nMol/kg) tration ments (mg/kg) 11 10Bevaciz- 10 mg/kg i.p. once 11 110 umab (68 nMol/kg) weekly 12 10 ANG2i-10 mg/kg i.p. once 6 60 LC06 (68 nMol/kg) weekly 13 10 ANG2i- 10 mg/kgi.p. once 6 60 LC06 + (68 nMol/kg) + weekly Bevaciz- 10 mg/kg i.p. once11 110 umab (68 nMol/kg) weekly 14 10 TvAb- 13 mg/kg i.p. once 6 782441- (64 nMol/kg) weekly bevaciz- umab- LC06

Monitoring:

Animals were controlled 2× per week for their health status. Bodyweights were documented 2× per week after cell injection. The tumordimensions were measured by caliper beginning on the staging day andsubsequently 2 times per week during the whole treatment period. Tumorvolume was calculated according to NCI protocol (Tumor weight=1/2ab²,where “a” and “b” are the long and the short diameters of the tumor,respectively). Termination criteria were the critical tumor mass (up to1.7 g or Ø1.5 cm), body weight loss more than 20% from baseline, tumorulceration or poor general condition of the animals.

Results: Tumor growth inhibition based on medians (in percent) at day 91

TGI ANG2i-LC06 10 mg/kg (68 nMol/kg) i.p.; 45.3 Bevacizumab 10 mg/kg (68nMol/kg) i.p. ANG2i-LC06 10 mg/kg i.p. (68 nMol/kg) 44.4TvAb-2441-bevacizumab-LC06_13 mg/kg i.p. 60.4 (64 nMol/kg)

The results show that the bispecific <VEGF-ANG-2> antibodyTvAb-2441-bevacizumab-LC06 showed a higher tumor growth inhibition (atlower doses) in the bevacizumab-resistant xenograft tumor model Colo205in Scid beige mice compared to the treatment with monospecific antibodyANG2i-LC06 alone or the combination of ANG2i-LC06 and bevacizumab.

Example 17 In Vivo Inhibition of Tumor Angiogenesis in s.c. Calu-3 NSCLCXenograft

Detection Via Non-Invasive In Vivo Imaging of Angiogenesis UsingAnti-CD31 Labeled with Cell Lines and Culture Conditions:

This human lung adenocarcinoma cancer cell line has been establishedfrom a human caucasian male with lung cancer. Cells were obtained fromRoche, Kamakura and passaged in house for working cell bank. Tumor cellsare routinely cultured in RPMI1640 medium (PAN Biotech, Germany)supplemented with 10% fetal bovine serum (PAN Biotech, Germany) and 2 mML-glutamine (PAN Biotech, Germany) at 37° C. in a water-saturatedatmosphere at 5% CO₂. Culture passage is performed with trypsin/EDTA 1×(PAN) splitting one time/week.

Animals:

Female BALB/c nude mice; age 4-5 weeks at arrival (purchased fromCharles River Germany) were maintained under specific-pathogen-freecondition with daily cycles of 12 h light/12 h darkness according tocommitted guidelines (GV-Solas; Felasa; TierschG). Experimental studyprotocol was reviewed and approved by local government. After arrivalanimals were maintained in the quarantine part of the animal facilityfor one week to get accustomed to the new environment and forobservation. Continuous health monitoring was carried out on regularbasis. Diet food (Provimi Kliba 3337) and water (acidified pH 2.5-3)were provided ad libitum. Age of mice at start of the study was about 10weeks.

Tumor Cell Injection:

At the day of injection, tumor cells were harvested (trypsin-EDTA) fromculture flasks (Greiner) and transferred into 50 ml culture medium,washed once and resuspended in PBS. After an additional washing stepwith PBS and filtration (cell strainer; Falcon 0100 μm) the final celltiter was adjusted to 5.0×10⁷/ml. Tumor cell suspension was carefullymixed with transfer pipette to avoid cell aggregation. After this, cellsuspension was filled into a 1.0 ml tuberculin syringe (Braun Melsungen)using a wide needle (1.10×40 mm); for injection needle size was changed(0.45×25 mm) and for every injection a new needle was used. Anesthesiawas performed using a Stephens inhalation unit for small animals withpreincubation chamber (plexiglas), individual mouse nose-mask (silicon)and not flammable or explosive anesthesia compound Isoflurane(cp-pharma) in a closed circulation system. Two days before injectioncoat of the animals were shaved and for cell injection skin ofanaesthetized animals was carefully lifted up with an anatomic forcepsand 100 μl cell suspension (=5.0×10⁶ cells) was injected subcutaneouslyin the right flank of the animals.

Treatment of Animals

At study day 35, mice were randomized to statistically well distributedgroups, depending on their body weight and tumor size. For the treatmentwith therapeutic antibodies, each group consisted of 10 mice andtreatment with therapeutic antibodies was applied once weekly i.p. for a6 week time period. (see FIG. 19)

-   Group 1: vehicle (Xolair) 10 mg/kg-   Group 2: Bevacizumab 10 mg/kg-   Group 3: Combination of monospecific <VEGF> bevacizumab 10 mg/kg    plus monospecific <ANG-2> Ang2i-LC06 10 mg/kg    (=bevacizumab/Ang2i-LC06)-   Group 4: Bispecific <VEGF-ANG-2> antibody 2441-bevacizumab-scFv-LC06    13.3 mg/kg

Monitoring:

Animals were controlled 2× per week for their health status. Bodyweights were documented 2× per week after cell injection. The tumordimensions were measured by caliper beginning on the staging day andsubsequently 2 times per week during the whole treatment period. Tumorvolume was calculated according to NCI protocol (Tumor weight=1/2ab²,where “a” and “b” are the long and the short diameters of the tumor,respectively). Termination criteria were the critical tumor mass (up to1.7 g or Ø>1.5 cm), body weight loss more than 20% from baseline, tumorulceration or poor general condition of the animals.

Blood Vessel and Angiogenesis Monitoring with Labeled Anti-CD 31Antibody

Preliminary studies revealed that anti-CD31 antibody as best agent forimaging tumor vasculature. This agent targets mouse endothelial CD31receptors and visualizes single blood vessels with a lowsignal-to-background ratio. Therefore, imaging for anti-CD31 antibodyrepresents a feasible way to image tumor vasculature. Three mice of eachtherapy group have been chosen and injected i.v. with 50 μg/mouseanti-CD3 antibody labeled covalently with the organic fluorophoreAlexa610 at day 35, 49 and 79. Near-infrared imaging was carried out 24hrs after each application of the labeled antibody under inhalationanesthesia. An increase or decrease of tumor vasculature was visualizedby using the compare image tool of the MAESTRO system. Under treatmentwith the control mab Xolair and the therapeutic antibody bevacizumab, anincrease of tumor blood vessels from day 35 to day 79 was observed. Incontrast, the combined treatment with bevacizumab plus Ang2i-LC06 and2441-bevacizumab-scFv-LC06 exhibited a decrease of tumor vasculature(FIG. 19).

Tumor regions were quantified by manually drawing measurement areas andsignal intensities were evaluated in intensity values (totalsignal/exposure time). The average changes of CD31 signals from day 35to 49 and from day 49 to 79 were plotted in FIG. 19. All treatmentgroups revealed an increase in tumor vasculature from day 35 to 49.While CD31 tumor signals steadily accelerated in group 1 (Xolair) andgroup 2 (bevacizumab), tumor vasculature significantly decreased ingroup 3 (Combination of bevacizumab plus <ANG-2> Ang2i-LC06) and group 4(Bispecific <VEGF-ANG-2> antibody 2441-bevacizumab-scFv-LC06), withgroup 4 showing clearly the most pronounced antiangiogenic effect (FIG.19).

Immediately after the last in vivo imaging studies, tumors wereexplantated (day 79), fixed in formalin and embedded in paraffin for exvivo studies. Fluorescence microscopy showed numerous well definedcapillaries in tumors treated with control mab Xolair. Several tumorblood vessels were observed in mice treated with bevacizumab. Incontrast, treatment groups 3 and 4 had significantly fewer and lessdefined blood vessels in the tumors compared to treatment groups 1 and 2whereas group 4 showed the most pronounced effect. Group 4 revealedlower microvessel density, capillaries were generally smaller andunstructured and they exhibited weaker anti-CD31 fluorescence signals asGroup 1, 2 and 3. Histochemical HE-staining showed intratumoral necroticregions for up to 90% of all regions in the treatment group with thebispecific antibody of group 4 which is clearly higher than for theother treatment groups (data not shown).

Example 18 In Vivo Efficacy of Bispecific Antibodies <VEGF-ANG-2> andCompared to the Parent Monospecific Antibodies (Alone or in Combination)in the Staged Subcutaneous Colo205 Xenograft Model in Scid Beige MiceCell Lines and Culture Conditions:

Colo205 human colorectal cancer cells were originally obtained from ATCCand after expansion deposited in the Roche Penzberg internal cell bank.Tumor cell line was routinely cultured in RPMI 1640 medium (PAA,Laboratories, Austria) supplemented with 10% fetal bovine serum (PAALaboratories, Austria) and 2 mM L-glutamine, at 37° C. in awater-saturated atmosphere at 5% CO₂. Passage 2-5 was used fortransplantation.

Animals:

Female SCID beige mice; age 4-5 weeks at arrival (purchased from CharlesRiver Germany) were maintained under specific-pathogen-free conditionwith daily cycles of 12 h light/12 h darkness according to committedguidelines (GV-Solas; Felasa; TierschG). Experimental study protocol wasreviewed and approved by local government. After arrival, animals weremaintained in the quarantine part of the animal facility for one week toget accustomed to the new environment and for observation. Continuoushealth monitoring was carried out on regular basis. Diet food (ProvimiKliba 3337) and water (acidified pH 2.5-3) were provided ad libitum. Ageof mice at start of the study was about 10 weeks.

Tumor Cell Injection:

At day of injection Colo205 cells were centrifuged, washed once andresuspended in PBS. After an additional washing with PBS cellconcentration and cell size were determined using a cell counter andanalyzer system (Vi-CELL, Beckman Coulter). For injection of Colo205cells, the final titer was adjusted to 5.0×10E7 cells/ml, viability ca.90%. Subsequently 100 μl of this suspension corresponding to 2.5*106cells per animal was injected s.c. into the right flank of the mice.

Treatment of animals started at day of randomisation, 16 days after celltransplantation (study Ang2_PZ_Colo205_009)) at a mean tumor volume of100 mm3, respectively.

Dose schedule of Study Ang2_PZ_Colo205_009:

No of Dose Route/Mode of animals Compound (mg/kg) administration 10Xolair 10 i.p. once weekly 10 <VEGF> 10 i.p. once weekly Bevacizumab 10< ANG-2> 10 i.p. once weekly Ang2i-LC06 10 Ang2i-LC06 + 10 i.p. onceweekly Bevacizumab 10 i.p. once weekly 10 <VEGF-ANG-2> 13.3 i.p. onceweekly TvAb-2441- bevacizumab- LC06 10 <VEGF-ANG-2> 20 i.p. once weeklyBevacizumab-LC06- CH1-CL 10 <VEGF-ANG-2> 16.6 i.p. once weekly scFAb-Bevacizumab-LC06- 2620

Monitoring:

Animals were controlled 2× per week for their health status. Bodyweights were documented 2× per week after cell injection. The tumordimensions were measured by caliper beginning on the staging day andsubsequently 2 times per week during the whole treatment period. Tumorvolume was calculated according to NCI protocol (Tumor weight=½ab²,where “a” and “b” are the long and the short diameters of the tumor,respectively). Termination criteria were the critical tumor mass (up to1.7 g or Ø>1.5 cm), body weight loss more than 20% from baseline, tumorulceration or poor general condition of the animals.

Results: Tumor Growth Inhibition (TGI) Based on Medians (in Percent) atDay 61

TGI <VEGF> Bevacizumab 66 < ANG-2> Ang2i-LC06 47 Ang2i-LC06 +Bevacizumab 78 <VEGF-ANG-2> TvAb-2441-bevacizumab-LC06 87 <VEGF-ANG-2>Bevacizumab-LC06-CH1-CL 92 <VEGF-ANG-2> scFAb-Bevacizumab-LC06-2620 86

The results show that all three bispecific <VEGF-ANG-2>bevacizumab-ANG2i-LC06 antibodies (all based on the bevacizumabsequences SEQ ID No: 7 and 8 and on ANG2i-LC06 sequences SEQ ID No: 52and 53) showed a higher tumor growth inhibition in xenograft tumor modelColo205 in Scid beige mice compared to the treatment with monospecificantibodies ANG2i-LC06 and bevacizumab alone or the combination ofANG2i-LC06 and bevacizumab.

Example 19 Expression & Purification and Properties of Bispecific<VEGF-ANG-2> Antibody Molecules scFAb-Bevacizumab-LC10-2620,scFab-Bevacizumab-LC10-2640 and scFab-Bevacizumab-LC10-2641,Bevacizumab-LC10-KiH-C-scFab, Bevacizumab-LC10-C-Fab-6CSS,Bevacizumab-LC10-CH1-CL, Bevacizumab-LC10-VH-VL andBevacizumab-LC10-VH-VL-SS, Bevacizumab-LC10-N-scFab andBevacizumab-LC10-N-scFabSS

By replacing the VH and VL domains of Ang2i-LC06 (SEQ ID No: 52 and 53)with the corresponding VH and VL domains of Ang2i-LC10 (SEQ ID No: 84and 85) and using the (apart from such replacement) analogous proceduresand sequences described in Example 10 to 14, the bispecific,<VEGF-ANG-2> antibody molecules scFAb-bevacizumab-LC10-2620,scFab-bevacizumab-LC10-2640 and scFab-bevacizumab-LC10-2641,bevacizumab-LC10-KiH-C-scFab, bevacizumab-LC10-C-Fab-6CSS,bevacizumab-LC10-CH1-CL, bevacizumab-LC10-VH-VL andbevacizumab-LC10-VH-VL-SS, bevacizumab-LC10-N-scFab andbevacizumab-LC10-N-scFabSS, all based on <VEGF> bevacizumab and <ANG-2>Ang2i-LC10 are expressed and purified. Binding affinities and other invitro properties are determined as described in the Examples above.

Example 20 In Vivo Efficacy of Bispecific Antibody <VEGF-ANG-2>Molecules scFAb-Bevacizumab-LC10-2620, scFab-Bevacizumab-LC10-2640 andscFab-Bevacizumab-LC10-2641, Bevacizumab-LC10-KiH-C-scFab,Bevacizumab-LC10-C-Fab-6CSS, Bevacizumab-LC10-CH1-CL,Bevacizumab-LC10-VH-VL and Bevacizumab-LC10-VH-VL-SS,Bevacizumab-LC10-N-scFab and Bevacizumab-LC10-N-scFabSS

In vivo efficacy of bispecific antibody <VEGF-ANG-2> moleculesscFAb-bevacizumab-LC10-2620, scFab-bevacizumab-LC10-2640 andscFab-bevacizumab-LC10-2641, bevacizumab-LC10-KiH-C-scFab,bevacizumab-LC10-C-Fab-6CSS, bevacizumab-LC10-CH1-CL,bevacizumab-LC10-VH-VL and bevacizumab-LC10-VH-VL-SS,bevacizumab-LC10-N-scFab and bevacizumab-LC10-N-scFabSS is determinedanalogously to the corresponding Examples above.

1. A method of treatment of a patient suffering from a disease ordisorder which is cancer or a vascular disease, said method comprisingthe step of administering a bispecific antibody to a patient in the needof such treatment, said bispecific antibody being one that bindsspecifically to human vascular endothelial growth factor (VEGF) andhuman angiopoietin-2 (ANG-2) and comprises a first antigen-binding sitethat specifically binds to human VEGF and a second antigen-binding sitethat specifically binds to human ANG-2, wherein: i) said antigen-bindingsites each comprise an antibody heavy chain variable domain and anantibody light chain variable domain; ii) said first antigen-bindingsite comprises in the heavy chain variable domain: a CDR3 region havingan amino acid sequence selected from the group consisting of: SEQ ID NO:1, SEQ ID NO: 9, SEQ ID NO: 17, and SEQ ID NO: 94; a CDR2 region havingan amino acid sequence selected from the group consisting of: SEQ ID NO:2, SEQ ID NO: 10, SEQ ID NO: 18, and SEQ ID NO: 95; and a CDR1 regionhaving an amino acid sequence selected from the group consisting of: SEQID NO:3, SEQ ID NO: 11, SEQ ID NO: 19, and SEQ ID NO: 96, and in thelight chain variable domain: a CDR3 region having an amino acid sequenceselected from the group consisting of: SEQ ID NO: 4, SEQ ID NO: 12, SEQID NO: 20, and SEQ ID NO: 97, a CDR2 region having an amino acidsequence selected from the group consisting of: SEQ ID NO:5, SEQ ID NO:13, SEQ ID NO: 21, and SEQ ID NO: 98; and a CDR1 region having an aminoacid sequence selected from the group consisting of: SEQ ID NO:6, SEQ IDNO: 14, SEQ ID NO: 22, and SEQ ID NO: 99; and iii) said secondantigen-binding site comprises in the heavy chain variable domain: aCDR3 region having an amino acid sequence selected from the groupconsisting of: SEQ ID NO: 25, SEQ ID NO: 38, SEQ ID NO: 46, SEQ ID NO:54, SEQ ID NO: 62, SEQ ID NO: 70, SEQ ID NO: 78, and SEQ ID NO: 86; aCDR2 region having an amino acid sequence selected from the groupconsisting of: SEQ ID NO: 26, SEQ ID NO: 39, SEQ ID NO: 47, SEQ ID NO:55, SEQ ID NO: 63, SEQ ID NO: 71, SEQ ID NO: 79, and SEQ ID NO: 87; anda CDR1 region having an amino acid sequence selected from the groupconsisting of: SEQ ID NO:27, SEQ ID NO: 40, SEQ ID NO: 48, SEQ ID NO:56, SEQ ID NO: 64, SEQ ID NO: 72, SEQ ID NO: 80, and SEQ ID NO: 88; andin the light chain variable domain: a CDR3 region having an amino acidsequence selected from the group consisting of: SEQ ID NO: 28, SEQ IDNO: 28 with the mutations T92L, H93Q and W94T, SEQ ID NO: 41, SEQ ID NO:49, SEQ ID NO: 57, SEQ ID NO: 65, SEQ ID NO: 73, SEQ ID NO: 81, and SEQID NO: 89; a CDR2 region having an amino acid sequence selected from thegroup consisting of: SEQ ID NO:29, SEQ ID NO: 42, SEQ ID NO: 50, SEQ IDNO: 58, SEQ ID NO: 66, SEQ ID NO: 74, SEQ ID NO: 82 and SEQ ID NO: 90;and a CDR1 region having an amino acid sequence selected from the groupconsisting of: SEQ ID NO:30, SEQ ID NO: 43, SEQ ID NO: 51, SEQ ID NO:59, SEQ ID NO: 67, SEQ ID NO: 75, SEQ ID NO: 83, and SEQ ID NO:
 91. 2. Amethod according to claim 1 wherein said disease or disorder is cancer.3. A method according to claim 1 wherein said disease or disorder is avascular disease.
 4. A method according to claim 1 wherein saidbispecific antibody is characterized in that said first antigen-bindingsite comprises, in the heavy chain variable domain, a CDR3 region of SEQID NO: 1, a CDR2 region of SEQ ID NO: 2, and a CDR1 region of SEQ IDNO:3, and, in the light chain variable domain, a CDR3 region of SEQ IDNO: 4, a CDR2 region of SEQ ID NO:5, and a CDR1 region of SEQ ID NO:6;and said second antigen-binding site comprises, in the heavy chainvariable domain, a CDR3 region of SEQ ID NO: 46, a CDR2 region of SEQ IDNO: 47, and a CDR1 region of SEQ ID NO: 48, and, in the light chainvariable domain, a CDR3 region of SEQ ID NO: 49, a CDR2 region of SEQ IDNO: 50, and a CDR1 region of SEQ ID NO:
 51. 5. A method according toclaim 4 wherein said bispecific antibody is characterized in that saidfirst antigen-binding site comprises, as the heavy chain variabledomain, SEQ ID NO: 7, and, as the light chain variable domain, SEQ IDNO: 8, and said second antigen-binding site comprises, as the heavychain variable domain, SEQ ID NO: 52, and, as the light chain variabledomain, SEQ ID NO:
 53. 6. A method according to claim 1 wherein saidbispecific antibody is characterized in that said first antigen-bindingsite comprises, in the heavy chain variable domain, a CDR3 region of SEQID NO: 1, a CDR2 region of SEQ ID NO: 2, and a CDR1 region of SEQ IDNO:3, and, in the light chain variable domain, a CDR3 region of SEQ IDNO: 4, a CDR2 region of SEQ ID NO:5, and a CDR1 region of SEQ ID NO:6;said second antigen-binding site comprises, in the heavy chain variabledomain, a CDR3 region of SEQ ID NO: 62, a CDR2 region of SEQ ID NO: 63,and a CDR1 region of SEQ ID NO: 64, and, in the light chain variabledomain, a CDR3 region of SEQ ID NO: 65, a CDR2 region of SEQ ID NO: 66,and a CDR1 region of SEQ ID NO:
 67. 7. A method according to claim 6wherein said bispecific antibody is characterized in that said firstantigen-binding site comprises, as the heavy chain variable domain, SEQID NO: 7, and, as the light chain variable domain, SEQ ID NO: 8; andsaid second antigen-binding site comprises, as the heavy chain variabledomain, SEQ ID NO: 68, and, as the light chain variable domain, SEQ IDNO:
 69. 8. A method according to claim 1 wherein said bispecificantibody is characterized in that said first antigen-binding sitecomprises, in the heavy chain variable domain, a CDR3 region of SEQ IDNO: 1, a CDR2 region of SEQ ID NO: 2, and a CDR1 region of SEQ ID NO:3,and, in the light chain variable domain, a CDR3 region of SEQ ID NO: 4,a CDR2 region of SEQ ID NO:5, and a CDR1 region of SEQ ID NO:6; saidsecond antigen-binding site comprises, in the heavy chain variabledomain, a CDR3 region of SEQ ID NO: 78, a CDR2 region of SEQ ID NO: 79,and a CDR1 region of SEQ ID NO: 80, and, in the light chain variabledomain, a CDR3 region of SEQ ID NO: 81, a CDR2 region of SEQ ID NO: 82,and a CDR1 region of SEQ ID NO:
 83. 9. A method according to claim 8wherein said bispecific antibody is characterized in that said firstantigen-binding site comprises, as the heavy chain variable domain, SEQID NO: 7, and, as the light chain variable domain, SEQ ID NO: 8; andsaid second antigen-binding site comprises, as the heavy chain variabledomain SEQ ID NO: 84, and, as the light chain variable domain, SEQ IDNO:
 85. 10. A method according to claim 1 wherein said bispecificantibody is characterized in that the ratio of the binding affinities Ku(antigen-binding site specific for VEGF)/K_(D) (antigen-binding sitespecific for ANG-2) is 1.0-10.0.
 11. A method according to claim 1wherein said bispecific antibody is characterized in that the secondantigen-binding site that specifically binds to human ANG-2 does notspecifically bind to human Angiopoetin 1 (ANG-1).
 12. A method accordingto claim 1 wherein said bispecific antibody is bivalent, trivalent ortetravalent.
 13. A method according to claim 1 wherein said bispecificantibody is characterized in that (A) said first antigen-binding sitecomprises, in the heavy chain variable domain, a CDR3 region of SEQ IDNO: 9, a CDR2 region of SEQ ID NO: 10, and a CDR1 region of SEQ ID NO:11, and, in the light chain variable domain, a CDR3 region of SEQ ID NO:12, a CDR2 region of SEQ ID NO: 13, and a CDR1 region of SEQ ID NO: 14;and (B) said second antigen-binding site comprises, in the heavy chainvariable domain, a CDR3 region of SEQ ID NO: 78, a CDR2 region of SEQ IDNO: 79, and a CDR1 region of SEQ ID NO: 80, and, in the light chainvariable domain, a CDR3 region of SEQ ID NO: 81, a CDR2 region of SEQ IDNO: 82, and a CDR1 region of SEQ ID NO:
 83. 14. A method according toclaim 13 wherein said bispecific antibody is characterized in that (A)said first antigen-binding site comprises, as the heavy chain variabledomain, SEQ ID NO: 15, and, as the light chain variable domain, SEQ IDNO: 16; and (B) said second antigen-binding site comprises, as the heavychain variable domain, SEQ ID NO: 84, and, as the light chain variabledomain, SEQ ID NO: 85.